WO2021048172A2 - Véhicule de distribution pour l'administration in situ d'agents pharmaceutiques - Google Patents

Véhicule de distribution pour l'administration in situ d'agents pharmaceutiques Download PDF

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WO2021048172A2
WO2021048172A2 PCT/EP2020/075152 EP2020075152W WO2021048172A2 WO 2021048172 A2 WO2021048172 A2 WO 2021048172A2 EP 2020075152 W EP2020075152 W EP 2020075152W WO 2021048172 A2 WO2021048172 A2 WO 2021048172A2
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delivery vehicle
bsh
api
item
host cell
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PCT/EP2020/075152
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English (en)
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WO2021048172A3 (fr
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Susanne Manuela GERMANN
Swee Chuang Lim HALLWYL
Jørgen Hansen
Javier Porcayo LOZA
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River Stone Biotech Aps
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Priority to US17/640,307 priority Critical patent/US20220331377A1/en
Publication of WO2021048172A2 publication Critical patent/WO2021048172A2/fr
Publication of WO2021048172A3 publication Critical patent/WO2021048172A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure pertains to vehicles capable of producing and delivering active pharmaceutical compounds in situ for preventing, treating and/or relieving a disease.
  • Background of the invention [0002] The background art in the field of the present invention includes WO2010124387 disclosing highly BSH active bacteria.
  • US2017360850 discloses certain probiotic strains of the species Saccharomyces boulardii, and Castagliuolo et al. (1999) discloses that a certain Saccharomyces boulardii protease inhibits the effects of Clostridioides difficile toxins A and B in human colonic mucosa.
  • WO2015168534 pertains to therapeutic treatment of gastrointestinal microbial imbalances through competitive microbe replacement.
  • Further art including CN106754843, CN106591272, CN106399284 and WO2017123592 pertains to bacteria expressing bile salt hydrolases, while others including CN106754445, CN106399284, and CN106754844 pertain to Pichia pastoris yeast producing a bile salt hydrolase enzyme.
  • Hudson et al., 2014 discloses a genetically engineered S. boulardii heterologously expressing a protein in the gastrointestinal tract of mice.
  • Hudson et al., 2014 discloses a genetically engineered S.
  • BSH refers to Bile Salt Hydrolase, which is an enzyme natively produced by intestinal microflora and is a selection criteria for probiotics (Begley et al., 2006), that catalyzes the deconjugation of a conjugated bile acid into an unconjugated bile acid.
  • Examples of this reaction include deconjugating glycocholic acid (GCA) or taurocholic acid (TCA) into cholic acid (CA); glycodeoxycholic acid (GDCA) or taurodeoxycholic acid (TDCA) into deoxycholic acid (DCA), and glycochenodeoxycholic acid (GCDCA) or taurochenodeoxycholic acid (TCDCA) into chenodeoxycholic acid (CDCA).
  • GCDCA glycochenodeoxycholic acid
  • TCDCA taurochenodeoxycholic acid
  • the present invention provides in a first aspect a delivery vehicle comprising a genetically modified microbial host cell comprising one or more heterologous polynucleotides encoding and producing a one or more enzyme active pharmaceutical ingredient (API), wherein the vehicle is suitable for administering to the mammal and wherein the modified microbial host cell is capable of producing and delivering the one or more enzyme API in situ of the location in the body of a mammal in need of preventing, treating and/or relieving a disease.
  • a delivery vehicle comprising a genetically modified microbial host cell comprising one or more heterologous polynucleotides encoding and producing a one or more enzyme active pharmaceutical ingredient (API), wherein the vehicle is suitable for administering to the mammal and wherein the modified microbial host cell is capable of producing and delivering the one or more enzyme API in situ of the location in the body of a mammal in need of preventing, treating and/or relieving a disease.
  • API active pharmaceutical ingredient
  • the invention provides a polynucleotide construct comprising a polynucleotide sequence encoding the API, operably linked to one or more control sequences.
  • the invention provides a cell culture, comprising the microbial cell of the invention. and a growth medium.
  • the invention provides a method for producing the cell culture of the invention comprising a) culturing the microbial cell of any preceding claim at conditions allowing growth of the microbial cell; and b) optionally recovering and/or isolating the cell culture.
  • the invention provides a fermentation liquid or composition comprising cell culture of the invention.
  • the invention provides a composition comprising the vehicle, and/or cell culture of the invention and one or more carriers, agents, additives and/or excipients.
  • the invention provides a pharmaceutical composition comprising the vehicle, microbial cell and/or cell culture of the invention and one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • the invention provides a method for preparing a pharmaceutical preparation comprising mixing the vehicle and/or cell culture of the invention with one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • the invention provides a method for treating a disease comprising administering the pharmaceutical preparation of the invention in an amount for the vehicle to produce and deliver in situ a therapeutically effective amount of the API.
  • Figures [0013] Figure 1. Deconjugation of six conjugated bile acids into their respective three deconjugated bile acids by S. boulardii yeast strains expressing selected codon-optimized BSH enzymes (Sb BSH , listed in Table 1) under aerob conditions in mineral media. The respective BSH enzymes were expressed in a genetic background where the yeast gene combination Sc_KEX2/Sc_BiP was overexpressed.
  • Example 1 Strains made in Example 1 were analyzed under screening conditions as described in Example 2 in Delft media, aerob, and 24 h of incubation with 10 mM BAM (bile acid mix, for details see Example 2).
  • BAM bile acid mix
  • Figure 2 Deconjugation of six conjugated bile acids by Sb BSH strains expressing codon- optimized B. ovatus BSH (Bo_BSH_co) with and without His-tag under aerob conditions in mineral media.
  • Bo_BSH_co was expressed in different genetic backgrounds where the yeast gene combinations Sc_KEX2/Sc_BiP/Sc_PDI, Sc_KEX2/Sc_BiP or Sc_KEX2/Sc_PDI were overexpressed, and where Bo_BSH_co was either non-tagged or His-tagged (+ His-tag).
  • Strains made in Example 3 and listed in Table 1 were analyzed under screening conditions as described in Example 2 in Delft media, aerob, and 24 h of incubation with 10 mM BAM.
  • Commercial Chologlycine hydrolase (CGH) from Clostridium perfingens (Sigma) was used as positive control for deconjugation. Error bars represent standard deviations between four independent biological clones of the initial screening.
  • Figure 3 Deconjugation of six conjugated bile acids by Sb BSH strains expressing codon- optimized B. ovatus BSH (Bo_BSH_co) under anaerob conditions in FeSSCoF media mimicking gastrointestinal conditions.
  • Bo_BSH_co was expressed in different genetic backgrounds where the yeast gene combinations Sc_KEX2/Sc_BiP/Sc_PDI, Sc_KEX2/Sc_BiP or Sc_KEX2/Sc_PDI were overexpressed.
  • Strains made in Example 1 and listed in Table 1 were analyzed under screening conditions as described in Example 2 in falcon tubes, FeSSCoF media, anaerob, and 72 h of incubation with 10 mM BAM.
  • FIG. 1 Deconjugation of six conjugated bile acids by Sb BSH strains listed in Table 1 expressing selected codon-optimized BSH enzymes in different media and with different conjugated bile acid substrates. The respective BSH enzymes were expressed in a genetic background where the yeast gene combination Sc_KEX2/Sc_BiP was overexpressed.
  • A) Strains made in Example 1 were analyzed under screening conditions as described in Example 5a in BHIS media, semi-anaerob, and 24 h of incubation with 10 mM BAM.
  • CGH Chologlycine hydrolase
  • Example 1 Strains made in Example 1 were analyzed under screening conditions as described in Example 5a in FeSSCoF media, semi-anaerob, and 24 h of incubation with 2% Ox Bile (Sigma Aldrich).
  • Ox Bile Sigma Aldrich
  • Figure 5 Aerobic growth curve of yeast strains. Panel A shows the growth of the wild type and five Sb BSH strains, and panel B shows the wild type and four other Sb BSH strains, all listed in Table 1.
  • FIG. 10 Inhibition of C. difficile spore germination by Sb BSH .
  • Supernatants from Sb BSH strains grown in FeSSCoF +/- 10 mM BAM were incubated for 15 min with 10 7 CD630 (Panel A) or R20291 (Panel B) spores in the presence of 1% TCA and plated on BHIS with/without 0.1% NaTa. Germination (%) is shown.
  • Figure 11 Growth profile of Sb BSH strains.
  • FIG. 12A Symptoms Group 1 (PBS, placebo). Percentage of animals displaying mild, moderate or severe symptoms in group 1. Deceased hamsters included. On day -3 animals were administered the appropriate Sb strain or PBS sham treatment as specified in Table 4, once per day (0.5 ml/dose).
  • CDI symptom grading was performed as described in Figure 11A.
  • Figure 12C Symptoms Group 3 (sSMT91, Sb BSH1 ). Percentage of animals displaying mild, moderate or severe symptoms in group 3. Deceased hamsters included. CDI symptom grading was performed as described in Figure 11A.
  • Figure 12D Symptoms Group 4 (sSMT95, Sb BSH2 ). Percentage of animals displaying mild, moderate or severe symptoms in group 4. Deceased hamsters included. CDI symptom grading was performed as described in Figure 11A.
  • Figure 13 Time from challenge to colonization. Colonization is defined by the presence of symptoms. Graphic representation of data shown in Table 5.
  • FIG. 5 Graphic representation of data shown in Table 5.
  • Figure 15. Kaplan-Meier survival analysis. Kaplan-Meier survival estimates after oral challenge of these animals with 100 spores of C. difficile 630.
  • Figure 16. C. difficile spores CFU in cecum (individual values). Upon death, hamsters were immediately frozen at -20 °C. On the day of analysis, hamsters were thawed, cecum removed and used for immediate C. difficile CFU analysis.
  • Figure 17. Toxin A levels in cecum (individual values). Upon death, hamsters were immediately frozen at -20 °C. On the day of analysis, hamsters were thawed, cecum removed and used for immediate toxin analysis by ELISA.
  • FIG. 1 S. boulardii CFU in feces (individual values). Once a hamster had succumbed to infection, feces were collected from the cage and analyzed for S. boulardii CFU. Incorporation by reference [0035] All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.
  • Any EC numbers used herein refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem.1994, 223, 1- 5; Eur. J. Biochem.1995, 232, 1-6; Eur. J. Biochem.1996, 237, 1-5; Eur. J. Biochem.1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively.
  • the nomenclature is regularly supplemented and updated; see e.g. http://enzyme.expasy.org/.
  • API or “Active Pharmaceutical Ingredient” as used interchangeable herein refers to any substance or mixture of substances intended to be used in the manufacture of a drug (medicinal) product and that, when used in the production of a drug, becomes an active ingredient of the drug product. Such substances are intended to furnish pharmacological activity or other direct and/or indirect effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or function of the body of humans or other animals.
  • active Ingredient refers to any component of a drug product intended to furnish pharmacological activity or other direct and/or indirect effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body of humans or other animals.
  • Active ingredients include those components of the product that may undergo chemical change during the manufacture of the drug product and be present in the drug product in a modified form intended to furnish the specified activity or effect.
  • the term API includes substances or mixtures of substances having an indirect pharmacological activity or other effect, by causing and/or activating another substance or mixture of substances to have a direct pharmacological activity or effect.
  • examples of such substances having indirect pharmacological activity or other effect include enzymes or enzyme co-factors promoting conversion of a substance or mixture of substances having less pharmacological activity into a substance or mixture of substances having more pharmacological activity.
  • a non-limiting example of an API considered herein is BSH.
  • BSH refers to Bile Salt Hydrolase, which is an enzyme catalyzing the deconjugation of a conjugated bile acid into an unconjugated bile acid.
  • deconjugation may be used interchangeably with the “bioconversion” of conjugated into unconjugated bile acid(s).
  • Examples of this reaction include deconjugating glycocholic acid (GCA) into cholic acid (CA); taurocholic acid (TCA) into cholic acid (CA); glycodeoxycholic acid (GDCA) into deoxycholic acid (DCA), taurodeoxycholic acid (TDCA) into deoxycholic acid (DCA), taurochenodeoxycholic acid (TCDCA) into chenodeoxycholic acid (CDCA), and glycochenodeoxycholic acid (GCDCA) into chenodeoxycholic acid (CDCA).
  • GCDCA glycochenodeoxycholic acid
  • a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell.
  • the terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.
  • the term "in vivo”, as used herein refers to within a living cell or organism, including, for example an animal, a plant or a microorganism.
  • in vitro refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.
  • in situ refers to an event that takes place locally, for example inside a living organism or cell, where the biological context is intact, for example where the cause or a symptom of a disease presents itself.
  • substrate or “precursor”, as used herein refers to any compound that can be converted into a different compound.
  • a conjugated bile acid can be a substrate for BSH and can be converted into a deconjugated bile acid.
  • substrates and/or precursors include both compounds generated in situ by an enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules that the host cell can metabolize into a desired compound.
  • Term "endogenous” or “native” as used herein refers to a gene or a polypepetide in a host cell which originates from the same host cell.
  • the term “deletion” as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.
  • the term “disruption” as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.
  • the term “attenuation” as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.
  • the terms “substantially” or “approximately” or “about”, as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed.
  • deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from.
  • the terms of degree can include a range of values plus or minus 10% from that value.
  • deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.
  • the term “and/or” as used herein is intended to represent an inclusive “or”.
  • the wording X and/or Y is meant to mean both X or Y and X and Y.
  • isolated refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but are not limited to compounds of the invention for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the invention may be isolated into a pure or substantially pure form.
  • a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process.
  • Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly.
  • the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.
  • non-naturally occurring refers to any substance that is not normally found in nature or natural biological systems. In this context the term “found in nature or in natural biological systems” does not include the finding of a substance in nature resulting from releasing the substance to nature by deliberate or accidental human intervention.
  • Non-naturally occurring substances may include substances completely or partially synthetized by human intervention and/or substances prepared by human modification of a natural substance.
  • % identity as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later.
  • the protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
  • BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences.
  • mature polypeptide or “mature enzyme” as used herein refers to a polypeptide in its final active form following translation and any post-translational modifications, such as N-terminal processing, endo- and exoproteolytic cleavage, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
  • cDNA refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, 3’-end processing and polyadenylation, before appearing as mature spliced mRNA.
  • coding sequence refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide.
  • control sequence refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide.
  • a control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide.
  • Control sequences include, but are not limited to promoter sequences, intronic splicing elements, and transcription terminator (stop) sequences on the DNA level, and leader sequences/5’ untranslated regions (5’UTR) and poly(A) signal sequences on the mRNA level. To be operational, control sequences usually must include promoter sequences and terminator sequences. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the region of a polynucleotide encoding a polypeptide. [0059] The term “signal peptide” as used herein refers to a polypeptide sequences that in some embodiments prompts a cell to translocate a protein to a location within or outside the cell.
  • pre-protein refers to a protein that has a signal sequence.
  • pro-protein refers to an inactive polypeptide that may be converted by the host into an active protein by proteolysis of an inhibitory sequence. A pre-pro-protein has both sequences still present.
  • N-terminal signal peptides such as pre-, pro-, or pre-pro sequences are required.
  • expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
  • expression vector refers to a DNA molecule, double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
  • Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and/or chromosomes comprising such genes.
  • microbial host cell refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Microbial host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • polynucleotide construct refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.
  • control sequence refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.
  • nucleotide sequence and polynucleotide are used herein interchangeably.
  • the term “comprise” and “include” as used throughout the specification and the accompanying claims as well as variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
  • the articles “a” and “an” are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article.
  • an element may mean one element or more than one element.
  • cell culture refers to a culture medium comprising a plurality of host cells of the invention.
  • a cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains.
  • the culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or monosodium glutamate; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.
  • a recombinant host e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium
  • additional components e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate
  • a nitrogen source such as ammonium sulfate, urea, or
  • Delivery vehicles for in situ API delivery are used interchangeably a system capable of delivering an active pharmaceutical ingredient.
  • the first aspect of the invention relates to a delivery vehicle comprising a system producing an active pharmaceutically ingredient (API) capable of preventing, treating and/or relieving one or more diseases in a mammal, wherein the vehicle is suitable for administering to the mammal and wherein the vehicle is capable of delivering the produced API in situ of the location in the mammal body in need of preventing, treating and/or relieving the disease.
  • the delivery vehicle of the invention can be any vehicle capable of hosting the system producing the API.
  • the system can include polynucleotides encoding the API and means for expressing such polynucleotides into the API.
  • the vehicle can comprise one or more microbial host cells comprising the one or more polynucleotides encoding the API.
  • the vehicle can be one or more microbial host cell, which optionally have been subjected to a protective encapsulation as described below.
  • such microbial host cell is genetically modified and particularly the one or more polynucleotides encoding the API is heterologous to the genetically modified cell.
  • the delivery vehicle comprises an extract of one or more microbial host cell cultures, said one or more microbial host cells comprising polynucleotides encoding the API and means for expressing such polynucleotides into the API.
  • the API of the invention can be an organic molecule particularly an organic molecule having a molecular weight of more than 200 g/mol, such as more than 500 g/mol, such as more than 1000 g/mol, such as more than 1500 g/mol; such as more than 2000 g/mol, such as more than 5000 g/mol.
  • the API can be a polypeptide, more particularly an enzyme.
  • the API is capable of in situ converting a substance or mixture of substances which is inactive in the preventing, treating and/or relieving one or more diseases into a compound which is active in the preventing, treating and/or relieving the one or more diseases.
  • the API is an enzyme that is secreted from a host cell.
  • the API is a BSH enzyme that is secreted out of a host cell and capable of performing a bile acid deconjugation reaction in the gastrointestinal tract of a subject to be treated.
  • the API is BSH active in the prevention, treatment and/or relief of infection by a pathogenic strain of Clostridioides difficile. [0074] Pathogenic strains of C.
  • TcdA and TcdB also called toxin A and toxin B
  • C. difficle virulence factors are the primary markers for diagnosis of CDI.
  • the API is a Bile Salt Hydrolase (EC 3.5.1.24) (BSH) comprising a polypeptide selected from: i) a polypeptide which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the mature BSH enzyme of anyone of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 111; ii)
  • the vehicle of the invention comprises one or more polynucleotides encoding the API, wherein the one or more polynucleotides are at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to any one of the polynucleotides comprised in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 or 112, or
  • the mature enzyme comprises the amino acids in positions 22 to 356, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 365, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 344, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 28 to 351, whereas the amino acids in positions 1 to 27 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 344, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 344, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 338, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 257, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 336, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 358, whereas the amino acids in positions 1 to 21 comprise a pre- signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 348, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 337, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 358, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 334, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 337, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 337, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 337, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 349, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 346, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 336, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 38 to 363, whereas the amino acids in positions 1 to 37 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 355, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 349, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 336, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 336, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 26 to 351, whereas the amino acids in positions 1 to 25 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 341, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 344, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 27 to 349, whereas the amino acids in positions 1 to 26 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to344, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 25 to 346, whereas the amino acids in positions 1 to 24 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 25 to 261, whereas the amino acids in positions 1 to 24 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 349, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 349, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 328, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 367, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 26 to 352, whereas the amino acids in positions 1 to 25 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 25 to 360, whereas the amino acids in positions 1 to 24 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 355, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 19 to 357, whereas the amino acids in positions 1 to 18 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 27 to 359, whereas the amino acids in positions 1 to 26 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 26 to 363, whereas the amino acids in positions 1 to 25 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 23 to 359, whereas the amino acids in positions 1 to 22 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 345, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 356, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 366, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • the mature enzyme comprises the amino acids in positions 22 to 350, whereas the amino acids in positions 1 to 21 comprise a pre-signal sequence.
  • such host cell can be any suitable host cell such as a fungus or a bacterium.
  • fungi may be selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia.
  • fungus is a yeast selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces.
  • yeast may be selected from the species consisting of Kluyveromyces lactis, Saccharomyces boulardii, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica.
  • the yeast is a Saccharomyces boulardii.
  • the Saccharomyces boulardii host cell may be a strain, variant or sub-type of the species Saccharomyces boulardii, referred to individually and collectively herein as an Sb strain.
  • the vehicle is a genetically modified yeast cell of the species S. boulardii secreting BSH, examples of which have been created for the first time and referred to herein as Sb BSH strain(s).
  • Sb BSH strain(s) examples of which have been created for the first time and referred to herein as Sb BSH strain(s).
  • fungal and in particular yeast cells are particularly preferred where the disease to be treated or prevented is a bacterial infection resistant to treatment with broad spectrum antibiotics, and the API is affecting these pathogenic bacteria in an inhibitory fashion. Since antibiotics do not have any effect on fungi, for example S.
  • bacterial host cells may be selected from the genera consisting of Lactobacillus, Leuconostoc, Streptomyces, Pediococcus, Lactococcus, Bifidobacterium, Weissella, Streptococcus, Komagataeibacter, Acetobacter, Escherichia, and Gluconacetobacter.
  • bacterial host cells may be selected from the species consisting of Escherichia coli, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paraplantarum, Lactobacillus coryniformis, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp.
  • Lactobacillus lactis Bifidobacterium bifidum, Leuconostoc mesenteroides, Leuconostoc citreum, Leuconostoc argentinum, Pediococcus pentosaceus, Weissella spp., Streptococcus thermophilus, Streptomycess spp., Gluconacetobacter xylinus, Acetobacter pasteurianus, Acetobacter aceti and Gluconobacter oxydans.
  • the microbial host cell of the invention further comprises at least one transporter molecule facilitating transport of the API and/ or any substrate or precursor required to produce the API in the vehicle.
  • the microbial host cell of the invention may also comprise one or more native or native codon-optimized genes which has been overexpressed, attenuated, disrupted and/or deleted, for example to enhance the production of the API and/or to stabilize the host cell.
  • the microbial host cell of the invention may comprise at least 2 copies or more of a polynucleotide encoding the API, or encoding one or more polypeptides comprising the operative protein production and/or secretion pathway.
  • the vehicle may comprise a microbial cell comprising the system producing the API and in particular, the vehicle is a microbial cell comprising the said system.
  • the vehicle is a genetically modified yeast strain, particularly of the genus Saccharomyces, more particularly of the species S. boulardii comprising and expressing a heterologous gene encoding a BSH enzyme and thereby producing and secreting said BSH enzyme.
  • the vehicle may comprise one or more recombinant microbial host cells capable of delivering one or more API to the location of a C. difficile infection or C. difficile- associated colitis.
  • the recombinant microbial host comprises more than one system for producing API and is capable of in situ production of a selection of APIs (such as a selection of BSH) considered to be most efficacious in the prevention or treatment of a certain condition (Jia et al., 2020 - Metagenomic analysis of the human microbiome reveals the association between the abundance of gut bile salt hydrolases and host health).
  • Formulations [0084] It is well understood to those skilled in the art that for API’s to be delivered for example to the gastrointestinal tract, it is desirable to protect such API’s from degradation in storage or in the body due to gastric juices, thermal and pH stresses, osmotic shock, and oxidative stress.
  • General drug delivery technologies for API’s often include encapsulation in enteric polymer coatings or capsules which can be triggered to release upon specific pH or osmotic conditions, in the presence of enzymes, or through time-release technologies. See for example https://www.capsugel.com/biopharmaceutical-technologies/enteric-drug-delivery-technologies); S. Amidon et al;. AAPS PharmSciTech. 2015 Aug; 16(4): 731–741; J. Li, D.J. Mooney; Nat. Rev. Mater. 2016 Dec; 1(12): 16071; H. Wen et al.; AAPS J. 2015 Nov; 17(6): 1327–1340; and L.
  • a common technique for microbial cells such as probiotics is encapsulation with for example gums, proteins, or alginate, and sometimes multi-layer coatings are used and can include materials such as starches, dextran sulfate and chitosan. Further, viability of Saccharomyces boulardii can be enhanced with encapsulation of layers of chitosan-dextran sulfate polyelectrolytes that are responsive to pH (Ben Thomas M et al.; J. Food Eng.; 2014; 136:1–8. [0085] Accordingly, in one embodiment the vehicle of the invention is coated by a protective coating.
  • the vehicle of the invention is encapsulated by a membrane, in a capsule, microcapsule, sphere and/or microsphere.
  • the coating, membrane, capsule, microcapsule, sphere and/or microsphere may in some embodiments be enteric, which is optionally triggered to release the vehicle and/or the API by pH, by osmotic pressure, by enzymatic digestion and/or by time-release.
  • the coating, capsule, microcapsule, sphere and/or microsphere of the invention may comprise one or more materials selected from gums, proteins, waxes, polyols, alginates, starches, dextrans and chitosans.
  • the coating, capsule, microcapsule, sphere and/or microsphere is insoluble in mammal gastro-intestinal juices.
  • the coating, capsule, microcapsule, sphere and/or microsphere is permeable to the API.
  • the vehicle comprises a microbail cell
  • the coating, capsule, microcapsule, sphere and/or microsphere is impermeable to the cell.
  • the coating, capsule, microcapsule, sphere and/or microsphere can be made of alginate-poly-L-lysine-alginate (APA).
  • the coating, capsule, microcapsule, sphere and/or microsphere can be made of materials selected from alginate/poly-L-lysine/pectin/poly-L-lysine/alginate (APPPA), alginate/poly-L-lysine/pectin/poly-L- lysine/pectin (APPPP), and alginate/poly-L-lysine/chitosan/poly-L-lysine/alginate (APCPA).
  • APPPA alginate/poly-L-lysine/pectin/poly-L-lysine/alginate
  • APPPP alginate/poly-L-lysine/pectin/poly-L- lysine/pectin
  • APIPA alginate/poly-L-lysine/chitosan/poly-L-lysine/alginate
  • the disease can be infections by pathogenic microorganisms such as bacteria of the genus of Clostridioides. It has been shown that broth of the BSH secreting bacteria Bacteroides ovatus inhibits proliferation of vegetative cells of Clostridioides difficile only in the presence of bile salts (Yoon et al., 2017), so, in another embodiment the pathogenic microorganism is Clostridioides difficile and the disease C. difficile infection (CDI) and/or C. difficile-induced colitis (CDAD, C. difficile-associated disease) as disclosed for example in: John S. Fordtran; Proc (Bayl Univ Med Cent); 2006 Jan; 19(1): 3–12.
  • CDI C. difficile infection
  • CDAD C. difficile-induced colitis
  • the infection to be treated may be by a pathogenic C. difficile strain with ribotype 027 as described by Tijerina-Rodriguez et al.; 2019, or any other pathogenic C. difficile strain with a different ribotype.
  • the infection can be non-recurrent C. difficile infection (NR-CDI) or recurrent C. difficile infection (R-CDI).
  • NR-CDI non-recurrent C. difficile infection
  • R-CDI recurrent C. difficile infection
  • high-fat diets increase bile acid levels in the gut.
  • High-level expression of BSH in recombinant mice has been seen to result in a significant reduction of conjugated bile acids, plasma cholesterol, liver triglycerides, and host weight gain.
  • BSH enzymes native to Bacteroidetes may ameliorate obesity (Jia et al., 2020). Indeed, the known correlation of more abundant gut bile salt hydrolases with improved specific aspecs of human health (summarized in Jia et al., 2020) is such that in some aspects diseases suitable for treatment by embodiments of the current invention comprising administration of a vehicle compring genetically modified microbial host cells expressing BSH include, but are not limited to, obesity, type 2 diabetes, cardiovascular disease, colon cancer, polycystic ovary syndrome, neurological diseases, diseases of the liver including nonalcoholic steatohepatitis, cirrhosis and/or liver cancer.
  • the API is BSH active in the prevention, treatment and/or relief of infection by a pathogenic strain of Clostridioides, such as C. difficile.
  • BSH as API may prevent, treat and/or relieve dysbiosis and/or diarrhea.
  • the API (such as BSH) may prevent, treat and/or relieve one or more diseases selected from a Clostridioides infection, Clostridioides infection induced colitis, obesity, type 2 diabetes, cardiovascular disease, colon cancer, polycystic ovary syndrome, a neurological disease, diseases of the liver including nonalcoholic steatohepatitis, cirrhosis and/or liver cancer.
  • the vehicle of the invention is a genetically modified S. boulardii host cell secreting BSH, active in the treatment of one or more diseases, particularly a C. difficile gut infection in a mammal, wherein the host cell is suitable for administration to the mammal and wherein the host cell is capable of producing and delivering the produced API(s) in situ at the specific location within the mammal’s body in need of preventing, treating and/or relieving the disease.
  • polynucleotide constructs and expression vectors [0093]
  • the invention also provides a polynucleotide construct comprising a polynucleotide sequence of the invention encoding a polypeptide of the operative metabolic pathway, operably linked to one or more control sequences, which direct the expression of the pathway polypeptide in a microbial host cell harboring the polynucleotide construct.
  • Conditions for the expression should be compatible with the control sequences.
  • one or more control sequences may be of a different source organism to the one or more polynucleotides whose expression they regulate.
  • the one or more polynucleotide sequence encoding the pathway polypeptide, or the control sequence, or both may be heterologous to the microbial host cell comprising the construct.
  • one or more polynucleotide sequences encoding a pathway polypeptide may be heterologous to the host organism, whilst one or more other polynucleotide sequences encoding other pathway polypeptides may be endogenous, homologous, or variants to polynucleotide sequences native to the host organism.
  • the polynucleotide construct is comprised in an expression vector. [0094] Polynucleotides may be manipulated in a variety of ways that allow expression of an API.
  • the control sequence may be a promoter, which is a polynucleotide that is recognized by the host cell for expression of a gene.
  • the promoter contains transcriptional control sequences that mediate the expression of the gene.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, synthetic, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • the promoter may also be an inducible promoter.
  • suitable promoters for directing transcription of the nucleic acid construct of the invention in fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral a-amylase, Aspergillus niger acid stable a-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus nidulans gpdA, Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, A.
  • NA2-tpi promoter is a modified promoter from an Aspergillus neutral a-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene.
  • promoters include modified promoters from an Aspergillus niger neutral a-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene.
  • Other examples of promoters are the promoters described in W02006/092396, W02005/100573 and W02008/098933, incorporated herein by reference.
  • promoters for directing transcription of the nucleic acid construct of the invention in a yeast host include promoters obtained from the genes for Saccharomyces cerevisiae translational elongation factor EF-1 alpha (TEF1, TEF2), S. cerevisiae fructose 1,6-bisphosphate aldolase (FBA1), S. cerevisiae glyceraldehyde-3-phosphate dehydrogenases (TDH1, TDH2, TDH3), S. cerevisiae enolase (EN01), S. cerevisiae galactokinase (GAL1), S.
  • TEF1 alpha TEF1 alpha
  • FBA1 S. cerevisiae fructose 1,6-bisphosphate aldolase
  • TDH1, TDH2, TDH3 S. cerevisiae enolase
  • EN01 S. cerevisiae galactokinase
  • GAL1 S. cerevisiae galactokina
  • yeast host cells include S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, or fusion promoter ADH2/GAP), S. cerevisiae triose phosphate isomerase (TPI1), S. cerevisiae metallothionein (CUP1), and S. cerevisiae 3-phosphoglycerate kinase (PGK1).
  • TPI1 trise phosphate isomerase
  • CUP1 S. cerevisiae metallothionein
  • PGK1 S. cerevisiae 3-phosphoglycerate kinase
  • Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488. Selecting a suitable promoter for expression in yeast is well known and is well understood by persons skilled in the art.
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3'-terminus of the gene encoding the polypeptide. Any terminator that is functional in the host cell may be used.
  • Useful terminators for fungal host cells can be obtained from the genes encoding Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger a-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease; while useful terminators for yeast host cells can be obtained from the genes for S. cerevisiae enolase (ENO1), S. cerevisiae cytochrome C (CYC1), S. cerevisiae alcohol dehydrogenase (ADH1), and S.
  • ENO1 S. cerevisiae enolase
  • CYC1 S. cerevisiae cytochrome C
  • ADH1 S. cerevisiae alcohol dehydrogenase
  • the control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • the control sequence may also be a 5 ⁇ untranslated region (5 ⁇ UTR, or leader sequence), a non-translated region of an mRNA that is important for translation by the host cell.
  • the leader sequence is operably linked to the 5'-terminus of the mRNA encoding the polypeptide. Any leader that is functional in the host cell may be used.
  • Useful leader sequences for fungal host cells can be obtained from the promoters of the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase, while useful leaders for yeast host cells can be obtained from the promoters of genes for S. cerevisiae enolase (EN01), S. cerevisiae 3-phosphoglycerate kinase (PGK1), and S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAPDH).
  • EN01 S. cerevisiae enolase
  • PGK1 S. cerevisiae 3-phosphoglycerate kinase
  • ADH2/GAPDH S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • the control sequence may also be a poly(A) signal sequence, which is a sequence operably linked to the 3'-terminus of the polynucleotide and that, during transcription, is recognized by the host cell RNA polymerase as a signal to add polyadenosine residues to the mRNA. Any poly(A) signal sequence that is functional in the host cell may be used.
  • Useful poly(A) signal sequences for fungal host cells can be obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger a-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease; while useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol.15: 5983-5990.
  • signal sequences may be present in the polynucleotides encoding polypetides so that the latter may undergo protein maturation and/or secretion.
  • polypeptides In order to enter the secretory pathway, polypeptides contain pre-, pro-, or pre-pro-peptide sequences that include a cleavage site that is recognized by signal peptidase in the ER lumen (reviewed in Barlowe et al., 2013). After entry into the ER, a polypeptide folds with or without the help of chaperones, and may undergo additional modifications such as disulfide bond formation, glycosylation, and oligomerization.
  • one or more polynucleotides encoding a polypeptide may comprise a suitable pre-, or pre-pro-sequence recognizable by the host cell.
  • bacterial pre-peptides are recognized in eukaryotes, this can be analyzed using the algorithm SignalP- 5.0 (http://www.cbs.dtu.dk/services/SignalP/).
  • BSH polypeptides derived from Gram-negative bacteria carry pre-peptides that possibly can be recognized in S. boulardii
  • BSH polypeptides derived from Gram-positive bacteria only carry a Methionine as start amino acid that is cleaved during protein processing in these bacteria, but has to be replaced with a pre-, or pre-pro-peptide that can be recognized in eukaryotes in order to enable protein secretion in S. boulardii.
  • regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Aspergillus niger glucoamylase promoter Aspergillus oryzae TAKA a-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used; while in yeast, the ADH2 system or GAL 1 system may be used.
  • RNA molecules in addition to the polynucleotide construct of the invention may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide sequence encoding the pathway polypeptide of the invention at such sites.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus or chromosomal) that can be conveniently subjected to recombinant DNA procedures and can cause expression of the gene encoding the pathway polypeptide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid (linear or closed circular plasmid), an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may, when introduced into the host cell, integrate into the host genome and replicate together with the chromosome(s) into which it has been integrated.
  • the vector may contain one or more selectable markers that permit convenient selection of transformed, transfected, transduced, or the like cells.
  • a selectable marker is a gene from which the product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophies, and the like.
  • Useful selectable markers for fungal host cells include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • Useful selectable auxotrophic markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • Useful drug resistance markers for yeast host cells include, but are not limited to, hph (hygromycin B phosphotransferase from Escherichia coli), ble (phleomycin resistance gene from Streptoalloteichus hindustanus), nat (nourseothricin N-acetyl transferase from Streptomyces noursei), and neo/kan (aminoglycoside 3'-phosphotransferase from transposon Tn903 giving resistance to G418).
  • the vector may further contain element(s) that permits integration of the vector into the genome of the host cell or permits autonomous replication of the vector in the cell independently of the genome.
  • the vector may rely on the polynucleotide encoding the pathway polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • yeast is the host, integration into the host genome is preferred by homologous recombination.
  • one or more additional “helper” vectors may be introduced into the host which contain additional polynucleotides encoding gene products for directing integration by homologous recombination into the genome of the host cell at precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as approximately 45 to approximately 2,000 base pairs, such as approximately 500 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell.
  • the integrational elements may be non-encoding or encoding polynucleotides.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any sequence mediating autonomous replication that functions in a cell.
  • the term "origin of replication" or "plasmid replicator” refers to a DNA sequence that enables a plasmid or vector to replicate in vivo.
  • Useful origins of replication for fungal cells include AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61- 67; Cullen et al., 1987, Nucleic Acids Res.
  • AMA1 sequence and construction of a plasmid comprising AMA1 can be accomplished using the methods disclosed in WO2000/24883.
  • Useful origins of replication for yeast host cells are the 2-micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
  • more than one copy of a polynucleotide encoding the pathway polypeptide of the invention may be inserted into a host cell to increase production of the API.
  • An increase in the copy number can be obtained by integrating one or more additional copies of the enzyme coding sequence into the host cell genome or by including a less efficient selectable marker gene on the plasmid, so that only cells containing multiple copies of the selectable marker gene - and thereby additional copies of the polynucleotide - can be selected by cultivating the cells in the presence of the appropriate selectable agent or media lacking the specific amino acid for auxotrophic growth.
  • the procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989; Mikkelsen et al., 2012; supra).
  • the vehicles of this disclose also include those comprising a microbial host cell comprising the polynucleotide construct as described, supra.
  • Cultures [0112]
  • the invention also provides a cell culture, comprising a host cell of the invention and a growth medium. Suitable growth mediums for host cells such as fungi and/or yeasts are known in the art. Methods of producing vehicles or cultures of the invention.
  • the invention also provides a method for producing the cell culture of the invention comprising a) culturing the microbial host cell of the invention at conditions allowing growth of the microbial host cell; and b) optionally recovering and/or isolating the cell culture.
  • the cell culture can be cultivated in a nutrient medium suitable for production of the compound of the invention and/or propagating cell count using methods known in the art.
  • the culture may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters in a suitable medium and under conditions allowing the host cells to grow and/or propagate, optionally to be recovered and/or isolated.
  • the cultivation can take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. from catalogues of the American Type Culture Collection).
  • a suitable nutrient medium comprise a carbon source (e.g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.), a nitrogen source (e. g. ammonium sulfate, ammonium nitrate, ammonium chloride, mono sodium glutamate etc.), an organic nitrogen source (e.g.
  • a carbon source e.g. glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, etc.
  • a nitrogen source e. g. ammonium sulfate, ammonium nitrate, ammonium chloride, mono sodium glutamate etc.
  • an organic nitrogen source e.g.
  • the cultivating of the host cell may be performed over a period of from about 0.5 to about 30 days.
  • the cultivation process may be a batch process, continuous or fed-batch process, suitably performed at a temperature in the range of 0-100 °C or 0-80 °C, for example, from about 0 °C to about 50 °C and/or at a pH, for example, from about 2 to about 10.
  • Preferred fermentation conditions for yeasts and filamentous fungi are a temperature in the range of from about 25 °C to about 55 °C and at a pH of from about 2 to about 9.
  • the method of the invention further comprises one or more elements selected from: a) culturing the cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of between 25 to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days.
  • the cell culture of the invention may be recovered and or isolated using methods known in the art.
  • the compound(s) may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, or lyophilization.
  • Fermentation composition [0118]
  • the invention further provides a fermentation composition comprising the cell culture of the invention.
  • the fermentation composition comprises an API produced by the culture and one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base (YNB), carbon source, and/or amino acids of the fermentation; wherein the concentration of the API is at least 1 mg/l composition.
  • the API concentration in the fermentation composition is at least 100 ⁇ g/L, 500 ⁇ g/L, 1 mg/L, 5 mg/L, such as at least 10 mg/L, such as at least 20 mg/l, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 500 mg/L, such as at least 1000 mg/L, such as at least 5000 mg/L, such as at least 10000 mg/L, such as at least 50000 mg/L.
  • the fermentation composition of the invention may have a cell density of at least 10 7 CFU/ml, such as at least 10 8 CFU/ml, such as at least 10 9 CFU/ml, such as at least 10 10 CFU/ml, such as at least 10 11 CFU/ml, such as at least 10 12 CFU/ml.
  • Compositions and use [0120]
  • the invention provides a composition comprising the vehicle, cell culture and/or fermentation composition of the invention and one or more carriers, agents, additives and/or excipients.
  • Carriers, agents, additives and/or excipients includes formulation additives, stabilising agent, fillers and the like.
  • the composition may be formulated into a dry solid form by using methods known in the art, such as spray drying, spray cooling, lyophilization, flash freezing, granulation, microgranulation, encapsulation or microencapsulation.
  • the composition may also be formulated into liquid stabilized form using methods known in the art, such as formulation into a stabilized liquid comprising one or more stabilizers such as sugars and/or polyols (e.g. sugar alcohols) and/or organic acids (e.g. lactic acid).
  • the composition is a pharmaceutical composition comprising the vehicle, cell culture and/or fermentation composition of the invention and one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • the pharmaceutical composition can be in form of a powder, tablet or capsule, or it can be liquid in the form of a pharmaceutical solution, suspension, lotion or ointment.
  • the invention further provides a method for preparing a pharmaceutical composition comprising mixing the vehicle, cell culture and/or fermentation composition of the invention with one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • the pharmaceutical composition is suitably used as a medicament in a method for treating a disease, in particular for preventing, treating and/or relieving CDI and/or CDI induced colitis.
  • the prevention, treatment and/or relief is in one embodiment inhibiting proliferation of a pathogenic strain of the genus Clostridioides, particularly by inhibition of Clostridioides spore germination as well as vegetative growth
  • the API is BSH and the treatment comprises contacting the pathogenic strain in the presence of a conjugated bile acids with the pharmaceutical composition at conditions allowing the vehicle, cell culture and/or fermentation composition to produce and deliver BSH in amounts converting the conjugated bile acids into therapeutically effective amounts of deconjugated bile acids inhibiting germination or proliferation of the pathogenic strain.
  • the pharmaceutical preparation and the treatment is performed in situ of the pathogenic strain in and/or on the body of a mammal.
  • the use and/or method of treatment of the invention can comprise administering the pharmaceutical composition of the invention in an amount for the vehicle, the cell culture and/or the fermentation composition of the invention to produce and deliver a therapeutically effective amount of the API, in situ, of the cause of the disease.
  • the use and/or method of treatment suitably includes administering the vehicle and or cell culture in an amount of at least 3 mg lyophilized cells, such as at least 10 mg, such as at least 25 mg such as at least 50 mg, such as at least 100 mg, such as at least 500 mg, per kg body mass per day to the mammal to be treated.
  • the treatment suitably includes administering the vehicle and or cell culture in the form of 1 or more dosages of an amount of at least 10 6 CFU, such as at least 10 7 CFU, such as at least 10 8 CFU, such as at least 10 9 CFU, such as at least 10 10 CFU, such as at least 10 11 CFU, such as at least 10 12 CFU per dose to the mammal to be treated.
  • the dosage regimen may have a frequency of doses of at least once, such as at least twice, such as least three times, such as at least four times, such as at least five times, such as at least six times daily.
  • the pharmaceutical preparation can be administered orally (preferably), or parenterally, such as topically, epicutaneously, sublingually, buccally, nasally, intradermally, intralesionally, (intra)ocularly, intramuscular, intrapulmonary and/or intravaginally.
  • the pharmaceutical composition can also be administered enterally to the gastrointestinal tract.
  • a composition suitable for oral administration comprises approximately 3-5x10 9 CFU/capsule in a plant cellulose capsule.
  • a composition suitable for oral administration comprises approximately 500 mg of lypholized host cells per capsule, said capsule comprising a body of gelatin and titanium dioxide, encapsulated by a coating comprising gelatin, titanium dioxide, red iron oxide and indigotin.
  • compositions suitable for oral administration comprising a cell culture, isolated recombinant host cells and/or fermentation composition of the invention may be administered after or simultaneously with one or more antibiotics.
  • suitable antibiotic treatments include vancomycin orally 4 times a day or fidaxomicin twice daily, both for a total of 10 days.
  • antibiotic treatment has contributed to disease onset (such as when treatment with broad spectrum antibiotics depletes the gut biome of the patient and is followed by C.
  • the recombinant host cells and/or fermentation composition of the invention are preferably yeast cells, more preferably Saccharomyces cells, most preferably Saccharomyces boulardii cells.
  • a suitable course of combined antibiotic and recombinant host cell treatment may be followed by the administration of one or more prebiotic polysaccharide(s) optionally in combination with supplementary doses of a cell culture, isolated recombinant host cells and/or fermentation composition of the invention.
  • prebiotics include but are not limited to fructooligosaccharides (FOS), inulins, galactooligosaccharides (GOS), resistant starch, pectin, beta-glucans and/or xylooligosaccharides.
  • FOS fructooligosaccharides
  • GOS galactooligosaccharides
  • resistant starch pectin
  • beta-glucans and/or xylooligosaccharides.
  • Example 1 Construction of Saccharomyces boulardii strains for production of BSH enzyme [0128] Kirkman (Kirkman Lab) and CNCM I-745 (Florastor®, Biocodex Inc) S. boulardii strains were modified to delete one or more of the native genes URA3, LEU2 and HIS3 in order to obtain auxotrophic mutants for the pathway insertion. URA3, LEU2 and HIS3 were deleted by removing the entire open reading frame (ORF) encoding the genes.
  • ORF open reading frame
  • the gRNA vectors were designed according to Vanegas et al.2017, and were constructed using uracil excision reaction-based cloning (Mikkelsen et al.2012).
  • the donor fragments were designed with sequences upstream and downstream of the ORF to be deleted and constructed by PCR.
  • S. boulardii Biocodex and S. boulardii Kirkman auxotrophic strains were further modified to express a selection of codon-optimized bile salt hydrolases (BSH) and codon-optimized S. cerevisiae genes that have been shown to improve protein production/secretion as disclosed in Hou et al.2012 and Huang et al.2018.
  • BSH codon-optimized bile salt hydrolases
  • Bacterial BSH sequences were analyzed and identified using SignalP-5.0 (Almagro Armenteros et al., 2019) to detect putative bacterial secretion peptides that can be recognized in eukaryotes. If no bacterial pre-peptide was recognized (which is the case in all genes sourced from Gram-positive bacteria), the first methionine was replaced by the B. ovatus BSH pre-peptide (MTKNLLLGIAAVCGSTFQAVA). [0129] An integration system, “Recombinator”, was used, which is similar to the S.
  • K. lactis LEU2 is available e.g. from EUROSCARF (http://www.euroscarf.de).
  • the integration system is build based on S. cerevisiae, so integration sites were used, which are highly conserved between S. cerevisiae and S. boulardii (Khatri et al., 2017). Genes were synthesized by Twist Bioscience, San Francisco, CA.
  • bZIP basic leucine zipper
  • the BSH genes were expressed using the well-known S. cerevisiae TDH3 promoter, Sc_KEX2_co/Sc_ERO1_co/Sc_SSO2_co/Sc_COG5_co were expressed using the S. cerevisiae TEF1 promoter, Sc_HAC1_co/Sc_SSO2_co were expressed using the S. cerevisiae TEF2 promoter, Sc_BiP_co/Sc_PDI_co were expressed using the S. cerevisiae PGK1 promoter, or Sc_BiP_co were expressed using the S.
  • boulardii transformants of Example 1 were tested in a deconjugation experiment in 96-well plates. All incubations were carried out at 30 °C with shaking at 230 rpm in a Kuhner Climo-Shaker ISF1-X. On day 0, a preculture of the BSH candidates was set up in 96 deep-well plates in 500 ⁇ L liquid synthetic complete medium lacking leucing (SC-leu) for 24 hours so all clones reach a steady state growth plateau in order to start out at a similar OD for the subsequent testing.
  • SC-leu liquid synthetic complete medium lacking leucing
  • S. boulardii is absent from the natural gut microbiota, however when administrated it has the capacity to colonize the colon (Blehaut et al.; 1989; Margret I Moré & Vandenplas; 2018; Margret Irmgard Moré & Swidsinski; 2015). For this reason, we tested BSH producers of S.
  • FeSSCoF fed-state simulated colonic fluid
  • InSitu TM Sb BSH cultures were then grown at 37 °C with shaking at 180 rpm for 3 days prior analysis, with 100 ⁇ l samples taken every 24 h.
  • the BSH enzyme was also catalytically active under unaerob conditions in gut like media ( Figure 3).
  • the combination of secretion boost genes only seems to have a minor effect on the deconjugation pattern, and the overall pattern and efficiency of deconjugation did not change discernably over time.
  • the recombinant S. boulardii strains of Table 1 were tested in a deconjugation experiment in 96-well plates.
  • the deconjugation reaction was incubated for up to 24 hours at 37 °C with shaking at 230 rpm under semi-anaerobic conditions, and supernatant was harvested by centrifuging the cultivation for 10 min at 4000g, and then diluting with water 1:200 for LC-MS measurement (see method below).
  • the deconjugation activity of the selected strains was working very well under anaerob conditions in FeSSCoF media ( Figure 4B (FeSSCoF, semi-anaerob)).
  • Example 3 Analysis of protein production, secretion and localization of bile salt hydrolase
  • BSH enzyme from Bacteroides ovatus with or without a functional His-tag in an auxotrophic Biocodex S. boulardii strain made in Example 1.
  • the bacterial BSH sequences were analyzed using SignalP-5.0 (Almagro Armenteros et al., 2019) for the detection of putative bacterial secretion peptides.
  • the Bacteroides ovatus pre-peptide was kept since it is recognized in Eukaryotes (Bo_bsh_co – SEQ ID NO: 2).
  • the strains expressing His-tagged Bo_bsh_co can be used to analyze protein localization in different cell fractions by Western blotting or by fluorescence microscopy of fixed cells.
  • Heterologous protein production, localization and secretion is analyzed in a similar way as described in (Wentz & Shusta, 2008) by inoculating the BSH expressing S. boulardii strains in liquid Mineral media, grown overnight at 37 °C with shaking at 230 rpm in a Kuhner Climo-Shaker ISF1-X prior to dilution to a uniform OD600 of 0.1 and regrowth for 3 days to an OD600 between 8 and 10.
  • Samples are taken by removing 100 ⁇ L of the culture for measurement of cell density (OD600), analysis of intracellular content, and determination of secretion yield. Protein levels are measured according to Wentz & Shusta, 2008 with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative Western blotting. Protein concentration is measured using Bradford Assay (Bio-Rad), using bovine serum albumin (BSA) as a standard. The cell-free BSH supernatants are resolved on a 12% polyacrylamide-SDS gel and transferred to nitrocellulose membranes.
  • the membranes are probed with the 6x-His Tag Monoclonal Antibody (3D5) antibody (1:1000; ThermoFisher Scientific) followed by a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (1:2000; Sigma, St. Louis, MO).
  • 3D5 6x-His Tag Monoclonal Antibody
  • HRP horseradish peroxidase
  • BSH enzyme activity is analyzed via three different assays with different sensitivity. a) BSH activity plate assay. [0139] The qualitative enzyme activity of the S.
  • boulardii strains with BSH is determined on solid media, derived from the CBH plate assay described in (Christiaens, Leer, Pouwels, & Verstraete, 1992). Strains are incubated at 30 °C for 72 h on synthetic complete (SC) solid media available e.g. from Sigma Aldrich containing 2% (w/v) glucose, 0.5% (w/v) TC (taurocholic acid, 5 g/L) or GC (glycocholic acid, 5 g/L), and 0.37 g/L CaCl2 under aerobic or anaerobic conditions.
  • SC synthetic complete
  • BSH assay for quantification of BSH activity on plate reader – Ninhydrin assay.
  • BSH activity is measured by determining the amount of amino acids released from conjugated bile salts as previously described (Liong & Shah, 2005), with several modifications described in (Jiang et al., 2010). Briefly, 4 ml stationary phase cells are centrifuged at 4,000 g and 4 °C for 20 min.
  • the cell pellet is then washed twice and resuspended in 3.5 ml 0.1 M phosphate buffer (pH 6.0). Next, 0.5 ml 2% sodium thioglycolate is added to the cell suspension and the mixture is sonicated for 4 min while constantly cooling on ice. The samples are then centrifuged at 13,000 g and 4 °C for 20 min. Next, 0.1 ml supernatant is mixed with 0.8 ml 0.1 M sodium phosphate buffer (pH 6.0) and 0.1 ml 50 mmol/l conjugated bile salt.
  • the mixture is then incubated at 37 °C for 30 min, after which 0.75 ml aliquots are mixed immediately with 0.75 ml 15% (w/v) trichloroacetic acid.
  • the samples were centrifuged at 13,000 g and 4 °C for 10 min, and then 1 ml of the supernatant is mixed with 2 ml ninhydrin reagent [0.5 ml 1% ninhydrin in 0.5 M citrate buffer (pH 5.5), 1.2 ml 30% glycerol, 0.2 ml 0.5 M citrate buffer pH 5.5).
  • the mixture is then boiled for 30 min and subsequently cooled for 3 min in ice water.
  • the absorption is recorded at 570 nm with a GloMax Discover microplate reader (Promega) and the amount of product formed is estimated from a calibration curve produced using glycine or taurine separately.
  • One unit of BSH activity is defined as the amount of enzyme that liberates 1 mmol amino acid from the substrate per minute.
  • the specific activity is defined as the number of units of activity per milligram of protein.
  • the protein concentrations of the supernatant are determined by the Bradford Agent (Bio-Rad) using bovine serum albumin as the standard. All experiments in this study are repeated three times.
  • the six major human bile salts are selected (Tanaka, Hashiba, Kok, & Mierau, 2000): taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and glycodeoxycholic acid (GDCA) obtained from Sigma.
  • TCA taurocholic acid
  • TCDCA taurochenodeoxycholic acid
  • TDCA taurodeoxycholic acid
  • GCDCA glycocholic acid
  • GCDCA glycochenodeoxycholic acid
  • GDCA glycodeoxycholic acid obtained from Sigma.
  • the rate of hydrolysis of conjugated bile salts is measured at 37 °C and at pH 6.5, which is similar to that of the small intestine in a healthy human (Corzo & Gilliland, 1999).
  • the released amount of amino acid from the substrates by enzymatic reaction is measured by ninhydrin assay and compared with the standard curve prepared by using either glycine or taurine. These experiments are conducted in triplicate.
  • the BSH activities are determined against the six major human conjugated bile salts according to (Hay & Carey, 1990; Staggers, Hernell, Stafford, & Carey, 1990): taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), and glycodeoxycholic acid (GDCA) obtained from Sigma.
  • TCA taurocholic acid
  • TCDCA taurochenodeoxycholic acid
  • TDCA taurodeoxycholic acid
  • GCDCA glycocholic acid
  • GCDCA glycochenodeoxycholic acid
  • GDCA glycodeoxycholic acid
  • Samples from Example 2 were prepared for LC-MS analysis by adjusting the pH to 7.5 with 10 mM NaOH to stop BSH activity. The samples were centrifuged at 4000 g in a microcentrifuge for 10 min, and for LC-MS quantification the supernatant was diluted 1:200 in water.
  • Targeted LC-MS analysis was performed to quantify conjugated and deconjugated bile acids. Liquid chromatography was performed on an Agilent 1290 Infinity II UHPLC with a binary pump and multisampler (Agilent Technologies, Palo Alto, CA, USA).
  • the liquid chromatography system was coupled to an Ultivo-Triple Quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray ion source (ESI) operated in positive and negative mode.
  • the Capillary voltage was maintained at 3500 V and the Nozzle voltage at 500 V.
  • Source gas temperature was set at 340 °C and source gas flow was set at 12 L/min.
  • Source sheath gas temperature was set at 380 °C and source sheath gas flow set at 12 L/min.
  • Nebulizing gas was set to 30 psi. Nitrogen used as a dry gas, nebulizing gas and collision gas.
  • Conjugated and deconjugated bile acids were detected in MRM mode in which the MRM transitions and mass spectrometer parameters (fragmentation voltage, collision energy, dwell time) were optimized for each metabolite.
  • Calculation of BSH activity is based on the release of deconjugated bile acid (CA, CDCA and DCA) from the hydrolysis of the amide bond of taurine- or glycine-conjugated bile acids.
  • the free bile salts are released linearly during the 24h incubation period.
  • Standard stock solutions of the six conjugated bile acids were made by dissolving 100 mM in 10 ml of water. The stock solutions were diluted in water to obtain a concentration of 10 mM.
  • C. difficile growth by recombinant Sb BSH culture supernatants (in vitro assay) a) Evaluation of growth of Sb BSH strains in different media and with different bile acid substrates [0144]
  • C. difficile is usually grown in the specific rich media BHIS (Brain Heart Infusion Supplemented) that is not typically used for culturing yeast, so in order to be sure that the Sb BSH strains exhibit satisfying deconjugation activity in this unusual media we conducted a deconjugation test of the strains in Table 1.
  • BHIS Brain Heart Infusion Supplemented
  • the BHIS used in this study had the following recipe: 37 g Brain heart infusion (Oxoid), 5 g Yeast extract (Oxoid), 1 g L-cysteine (Sigma-Aldrich), and optional 1000 ⁇ l of 1000x Resazurin (1 mg/ml in dH2O, Sigma-Aldrich).
  • the Sb BSH strains listed in Table 1 were grown as described in Example 2 in either FeSSCoF or BHIS media in high throughput formate.
  • Sb BSH strains displayed comparable deconjugation activity with 2% ox bile as in FeSSCoF media with BAM as substrate ( Figure 4C, compare to Figure 4B).
  • b) Sb BSH strain fitness [0147] To assess strain fitness, growth curves of strains listed in Table 2 were generated. Table 2: Selection of Sb BSH strains tested in in vitro assay
  • C. difficile strains can be used: 630 [tcdA + tcdB + , ribotype 012] (Wüst et al., 1982, ST11 [ribotype 078] (Knight et al., 2019), R20291 [ribotype 027] (Valiente et al., 2014), 90556-M6S [ribotype 001] (Type strain 9689, American Type Culture Collection (ATCC)), VPI 10463 [ribotype 087] (strain 43255, ATCC), M68 [ribotype 017] (Drudy, Harnedy, Fanning, O’Mahony, & Kyne, 2007; Lawley et al., 2009), isolate BI17-6443 of strain NAP1/BI/027 [ribotype 027] (Chen et al.; 2008
  • C. difficile (CD) strains were used: CD630 and R20291.
  • Overnight S. boulardii cultures in 3 ml BHIS media were set up in 14 ml culture tubes and incubated aerobically at 37 °C with shaking at 250 rpm. The next day, 500 ⁇ l of these yeast cultures were diluted in 14 ml culture tubes in 5 ml BHIS media containing 0mM bile acid mix (BAM), i.e., no supplements. 120 ⁇ l of these yeast cultures were also diluted in deep well plates in 1.2 ml BHIS media containing 0.5x (5mM) or 1x (10mM) BAM.
  • 5mM 0.5x
  • 10mM 10mM
  • CD cultures were then inoculated into 200 ml fresh BHIS broth at a 1:100 ratio in a deep-well 96-well plate; subsequently 200 ml of yeast supernatant obtained after 3 days of cultivation in BHIS media containing 0mM, 5mM or 10mM BAM (from -20 °C) were added to the CD cultures. Vegetative cell growth of CD was assessed after anaerobic incubation at 37 °C for 24h, samples were taken at time points 0, 6h and 24h. OD600 was measured using a microplate reader. The blank was 200 ml of BHIS broth, and control wells included BAM (5mM and 10mM) to control for its effect on CD growth.
  • C. difficile strains can be used: 630 [tcdA + tcdB + , ribotype 012] (Wüst et al., 1982, ST11 [ribotype 078] (Knight et al., 2019), R20291 [ribotype 027] (Valiente et al., 2014), 90556-M6S [ribotype 001] (Type strain 9689, American Type Culture Collection (ATCC)), VPI 10463 [ribotype 087] (strain 43255, ATCC), M68 [ribotype 017] (Drudy, Harnedy, Fanning, O’Mahony, and Kyne, 2007; Lawley et al., 2009), isolate BI17-6443 of strain NAP1/BI/027 [ribotype 027] (Chen et al.; 2008;
  • Cholestyramine resin (Sigma-Aldrich) was added to yeast supernatants supplemented with 0% and 1% bile acid to a final concentration of 50 mg/ml. The mixture was rocked at room temperature (RT) for 1 h, centrifuged, filtered through a 0.22 mm pore size filter (Advantec), and stored at -20 °C until use. [0157] CD growth was measured by determining OD600 after 24h of anaerobic incubation at 37 °C in the presence of the cholestyramine-treated yeast supernatant. Samples were taken at time points 0, 6h and 24h.
  • the blank was 200 ml of BHIS broth, and the effect of Ox bile on CD growth was also controlled for. Each reaction was tested in duplicate (200 ml measurements). All yeast strains listed in Table 2 were supplemented with 0 or 1% Ox bile during growth, and the supernatants underwent cholestyramine treatment (50 mg/ml) for an hour. Cholestyramine treated and untreated supernatants (0 or 1% Ox bile) were then tested for their inhibitory activity against CD630 and R20291. Samples were taken at 6h and 24h to measure the OD600 of CD in the presence of the supernatants.
  • yeast supernatant obtained as follows: Overnight S. boulardii cultures of strains listed in Table 2 were set up in 14 ml culture tubes in 3 ml BHIS media and incubated aerobically at 37 °C with shaking at 250 rpm. The next day, these yeast cultures were pelleted for 5 min at 4000 g and diluted in 14 ml culture tubes in 5 ml fed-state simulated colonic fluid (FeSSCoF) media containing 0 mM BAM.
  • FeSSCoF fed-state simulated colonic fluid
  • spores were serially diluted and plated on BHIS agar with or without supplementation of 0.1% (v/v) TCA. After overnight anaerobic growth at 37 °C, colonies were enumerated.
  • spores incubated in 100 ml of BHIS with 1% TCA (v/v) for 15 min were plated on BHIS containing 0.1% TCA (v/v) or on BHIS agar alone, respectively.
  • Germination rate (%) was calculated using the following formula: BHIS count / BHIS with 0.1% TCA x 100.
  • the BHIS count would be expected to include only vegetative cells while the BHIS containing 0.1% (w/v) TCA would include vegetative cells and spores.
  • Sb BSH strains could inhibit CD spore germination, supernatants from strains grown in either 0 or 10 mM BAM supplemented FeSSCoF medium were incubated with CD spores in the presence of 1% taurocholic acid ( Figure 10). The reactions were serially diluted and then plated on BHIS plates for vegetative cell counts, or on BHISS for total counts, and the germination rate was calculated.
  • Yeast strains sSMT88, sSMT91, and sSMT95 were re-activated from glycerol stocks on YPD agar plates. Then, the plates were incubated at 30 °C for two days. Subsequently, single colonies were inocculated in 5mL of YPD and incubated overnight at 30 °C and 180 rpm. These cultures were used as a seed to inoculate 50 mL of complex media contained in 250 ml shake flasks. We evaluated the growth of the strains in YPD and YPD/2x glucose (YPD_2x_glu).
  • Example 8 Formulation of recombinant S. boulardii strains as microsphere for GI delivery
  • the S. boulardii strains as described in Example 1 are washed in sodium phosphate buffer pH 6.8 and suspended at a ratio of 75% water and 25% cells and lactose to a final cell density of 3-9 X 10 10 CFU/gram and 4 g/L lactose.10 - 20 g/L of sodium alginate in sterile water are added at a ratio of 100 mL alginate solution to 10 - 40 g of the yeast suspension.
  • CDI Crohn's disease
  • CD630 is a highly infectious strain of C. difficile (Goulding et al., 2009). Spores of CD630 were prepared previously and purified through a 20- 50% Histodenz gradient (Phetcharaburanin et al., 2014) and stored at 4 o C. The spores were pre- validated to ensure infectivity. Preparation of S. boulardii for oral gavage administration [0166] 2-3 days prior to in vivo study, fresh S. boulardii cells were prepared by streaking the S. boulardii strains (sSMT88, sSMT91, sSMT95) onto YPD agar plates.
  • S. boulardii cell cultures were prepared by inoculating a full loop of cells into 48 ml of Yeast Extract–Peptone–Dextrose (YPD) liquid media in 250 ml baffled Erlenmeyer flasks. Culture flasks with Sb were incubated aerobically at 37°C with shaking, overnight, at 200 rpm. CFU /ml for each strain was determined and 6 ml was found to be sufficient for oral gavage of one animal. The next day, the overnight culture of each Sb strain was centrifugated at 4,000 rpm for 5 minutes.
  • YPD Yeast Extract–Peptone–Dextrose
  • boulardii strain in 500 ⁇ L 1x PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 ) were administered by oral gavage (Day -3 to end of study).24 hamsters were divided into 4 groups (1, 2, 3, 4) with 6 animals per group, listed in Table 3. Starting on day -3 and for the remainder of the study, animals received one daily dose of 1x PBS (Group 1, infection control), one daily dose of S. boulardii wild type (Group 2, Sb WT ), or one daily dose of the two best performing S. boulardii strains producing BSH of Example 6 (Group 3 and 4, Sb BSH1 and Sb BSH2 ).
  • Model Acute hamster model with CD630 challenge 20-hours post clindamycin administration (Hong et al., 2017a and b) Species: Hamsters, Golden Syrian.
  • Test study was conducted as follows: Day -3 to study end Animals housed individually in IVC chambers (1 animal/cage) Intra-gastric dosing of Sb (1 dose/day, 1-3 x 10 9 CFU in PBS, 0.5ml/dose) Intra-gastric dosing of Sb (as stated in Table 3) maintained daily (1 dose/day, ⁇ 1-3 x 10 9 CFU in PBS, 0.5ml/dose) Day -1 Animals administered a single dose of clindamycin-2-phosphate (30 mg/kg body weight) by oral gavage (intra-gastric gavage) Day 0: Animals administered a single oral (intra-gastric gavage) dose of CD630 spores (100 spores) Days 0-2 Monitoring of symptoms (last hamster succumbed to CDI at 50h) [0172] Sample analysis.
  • CFU C. difficile spore counts
  • ChromID agar BioMerieux
  • Collect content of cecum and colon Store at -20 °C for LC-MS analysis of bile acid pool, performed by Alderley Analytical Ltd, UK • Levels of toxins A in cecum (determined by ELISA) • % animals colonized with CD630 • Time between challenge and colonization • Graded and temporal assessment of symptoms prior to endpoint • Monitor size of appendix, harvest if visibly enlarged Correlates of retention and survival of Sb WT and Sb BSH to be determined: CFU enumeration of S.
  • boulardii suspension or PBS sham treatment as specified in Table 3, once per day (0.5 ml/dose). This was repeated daily until the end of the experiment on day 3 (50 hours).
  • clindamycin 2-phosphate was given to hamsters by single i.g. gavage at 30mg/kg.
  • Adverse effects and/or CDI symptoms were recorded daily from day -3. Upon death, hamsters were immediately frozen at -20 °C. Once a hamster had succumbed to infection, feces was collected from the cage and tested for S. boulardii CFU. On the day of analysis, hamsters were thawed, cecum removed and used for immediate toxin and CFU analysis.
  • ELISA analysis of toxin A was performed in order to assess disease progression as described previously in Hong et al., 2017a.
  • ELISA plates (GREINER, high binding type) were coated with Rabbit – anti-ToxA (1/6.000) in 0.01M PBS buffer pH 7.4, 50 ⁇ l per well. Plates were incubated overnight at RT, and washed 3x with PBS + 0.05% Tween20 (PBS+Tween). Blocking was performed with 2% BSA in PBS (150 ⁇ l/well) for 1hr at 37 °C, with 3 subsequent short washes with PBS+Tween.
  • CFU C. difficile spore counts
  • CFU S. boulardii counts
  • ELISA plates (GREINER, high binding type) were coated with Rabbit anti-ToxA (1/6.000) in 0.01M PBS buffer pH 7.4, 50 ⁇ l per well. Plates were incubated overnight at RT and washed three times with PBS + 0.05% Tween20 (PBS+Tween). Blocking was performed with 2% BSA in PBS (150 ⁇ l/well) for 1h at 37 °C, with three subsequent short washes with PBA+Tween. Samples were added (50 ⁇ l/well), starting with one half sample dilution of caecal extraction in diluent. After an incubation for 2 h at RT, plates were washed three times with PBS+Tween, with 1 min in between.
  • mouse anti-CDTA14 (1/1000) in diluent, 50 ⁇ l/well. After an incubation for 1 h at 30 °C, plates were washed three times, with two minutes interval.
  • anti-mouse HRP antibody Biolegend
  • the substrate Biolegend was added and the raction stopped with 2N H 2 SO 4 and the plate was read at 450nm.
  • Caecal pathology is scored in a blinded fashion, grading neutrophil margination (0, no neutrophil accumulation; 1, local acute neutrophil accumulation; 2, extensive submucosal neutrophil accumulation; 3, transmural neutrophilic infiltrate), haemorrhagic congestion (0, normal tissue; 1, engorged mucosal capillaries; 2, submucosal congestion with unclotted blood; 3, transmural congestion with unclotted blood), hyperplasia (0, no epithelial hyperplasia; 1, twofold increase in thickness; 2, threefold increase in thickness; 3, fourfold or greater increase in thickness), and percent of epithelial barrier involvement (0, no damage; 1, less than 10% of mucosal barrier involved; 2, less than 50% of mucosal barrier involved; 3, more than 50% mucosal barrier involved).
  • Results are expressed as mean pathology score per strain for each criterion.
  • Statistical analysis [0185] Graphs and statistics were generated using the Prism software (GraphPad Software, version 6.1). Survival data were analyzed by Kaplan–Meier survival analysis. Significance between treatment groups was calculated using the Post-hoc Power analysis (https://clincalc.com/stats/Power.aspx.). Results of the hamster model of CDI with Sb BSH [0186] The following results were obtained from the in vivo hamster CDI model. [0187] Observed hamster symptoms.
  • CDI hamsters often develop diarrhoea due to haemorrhagic caecitis which manifests as a ‘wet tail’, the diameter of which can indicate the severity and progression of disease (Buckley et al., 2011).
  • the CDI symptom grading of animals is subjective and the following symptom details were used as a guideline.
  • CDI symptoms were scored and grouped into three distinct categories. Mild diarrhoea and a wet tail (diameter less than 2 cm; i.e. the diameter of the visible external wet area on the hamster’s underbelly) were considered to be mild symptoms (+).
  • Group 2 (sSMT88) began to show symptoms at 34 hours post-challenge and by 38 hours, 4 hamsters had succumbed to CDI (67%) and the remaining 2 hamsters were showing symptoms. By 40 hours post- challenge all hamsters in group 2 had succumbed to CDI, demonstrating a slightly delayed infection time course compared to the placebo group. Both group 3 (sSMT91) and group 4 (sSMT95) showed a delayed time course compared to groups 1 and 2. The sSMT91 group began to show symptoms at 37 hours post-challenge, and by 41 hours 5 hamsters had succumbed to CDI (83%) with the remaining hamster surviving until 50 hours post challenge.
  • the sSMT95 group started to develop symptoms at 37 hours post-challenge and all hamsters were deceased by 41 hours.
  • the time course in infection for groups 3 and 4 were similar to one another. There was a noticeable difference in disease progression and symptom presentation between the placebo group (group 1) and sSMT88 (group 2, SbWT, wild type). There was a further delay in symptom presentation and progression between these 2 groups and the two treatment groups, sSMT91 (group 3, SbBSH1) and sSMT95 (group 4, SbBSH2).
  • Table 4 Observed hamster symptoms until 50 hours post-challenge (average values). 1 Post-hoc power was calculated using the website https://clincalc.com/stats/Power.aspx.
  • Table 5 Observed hamster symptoms until 41 hours post-challenge 1 (individual values).
  • RCDD recurrent C. difficile disease
  • the hamster model of CDI is adapted from Hong et al., 2017, Freeman et al., 2005, and Sambol, Tang, Merrigan, Johnson, & Gerding, 2001 with the following alterations.
  • a single dose of 30 mg/kg body weight clindamycin (clindamycin 2-phosphate, Sigma) is administered by oral gavage (Day -1)
  • individual doses of 2 mg/L vancomycin (vancomycin hydrochloride, Sigma) are administered by oral gavage (Day 1 to end of study)
  • individual doses of 1-3 x 10 9 CFU of respective recombinant Sb BSH strain in 500 ⁇ L 1x PBS are administered by oral gavage (Day -3 to end of study).
  • 30 hamsters are divided into 5 groups (1, 2, 3, 4, 5) with 6 animals per group, listed in Table 6.
  • animals receive one daily dose of 1x PBS (Group 1, infection control), one daily dose of S. boulardii wild type (Group 2, Sb WT ), one daily dose of the best performing S. boulardii strain producing BSH of Example 6 (Group 3, Sb BSH ), one daily dose of vancomycin (Group 4, vancomycin), or one daily dose of Sb BSH and vancomycin (Group 5, Sb BSH +vancomycin).
  • 1x PBS Group 1, infection control
  • S. boulardii wild type Group 2, Sb WT
  • Sb BSH best performing S. boulardii strain producing BSH of Example 6
  • vancomycin Group 4
  • vancomycin vancomycin
  • Sb BSH +vancomycin Group 5
  • all groups are administered clindamycin.
  • all hamsters are infected with 100 C. difficle spores of the CD630 strain by oral gavage 16h after clindamycin challenge.
  • C. difficile enumeration to analyse colonization efficiency Enumeration of the total C. difficile spore counts in cecum samples is performed as described by Lawley, et al. (2009). Thereby, the colonization efficiency of C. difficile is determined.
  • S. boulardii enumeration Enumeration of the total viable S. boulardii in feces is performed as described in (L. A. Chen et al., 2015) and (Toothaker & Elmer, 1984) with the following adjustments.
  • the feces are collected from cage at time of death. Each sample is dissolved by adding 5 mL 0.9 M saline solution with occasional vortexing for 1-2 h. A ten-fold serial dilution series corresponding to 10 10 CFU/mL to 0 CFU/mL is tested per experiment. The number of S. boulardii CFU in feces is determined by plating the serial dilutions on two sets of solid Sabouraud plates. Set A contains cycloheximide (0.5 g/L), and both sets A and B contain gentamicin (80 mg/L). Set B supports the unselective growth of various yeast species, including S. boulardii. Set A is selective for interfering Candida species.
  • Colonies are counted after incubation in an aerobic incubator at 37 °C for 2-3 days. For each experiment the difference in colony counts between the two plate sets is used to calculate the CFU spiked into each sample.
  • Bioanalysis Analytical quantification of the BSH substrate (conjugated bile salts) and conversion product (deconjugated bile salts) levels present in the colon and caecum is carried out by LC-MS as described above, and performed at Alderley Analytical, UK.
  • Histopathology [0206] Caecum samples are prepared for simple histology as described by (Buckley et al., 2013; Goulding et al., 2009) to evaluate mucosal damage and inflammation induced by the C. difficile toxins.
  • Caecal pathology is scored in a blinded fashion, grading neutrophil margination (0, no neutrophil accumulation; 1, local acute neutrophil accumulation; 2, extensive submucosal neutrophil accumulation; 3, transmural neutrophilic infiltrate), haemorrhagic congestion (0, normal tissue; 1, engorged mucosal capillaries; 2, submucosal congestion with unclotted blood; 3, transmural congestion with unclotted blood), hyperplasia (0, no epithelial hyperplasia; 1, twofold increase in thickness; 2, threefold increase in thickness; 3, fourfold or greater increase in thickness), and percent of epithelial barrier involvement (0, no damage; 1, less than 10% of mucosal barrier involved; 2, less than 50% of mucosal barrier involved; 3, more than 50% mucosal barrier involved).
  • Results are expressed as mean pathology score per strain for each criterion.
  • Statistical analysis [0207] Graphs and statistics are generated using Microsoft Excel or Prism software (GraphPad Software, version 6.1). Survival data are analyzed by Kaplan–Meier survival analysis. Significance between treatment groups is calculated using the Post-hoc Power analysis (https://clincalc.com/stats/Power.aspx.). Results [0208] The following results are obtainable from the in vivo hamster CDI model experiments. Table 7: Experimental outcomes of the hamster model for CDI and combined therapy with Sb BSH and antibiotic. Results from analyses performed on samples collected from the hamster model of CDI. [0209] C. difficile spore counts. C.
  • S. boulardii enumeration S. boulardii in vegetative form in faecal samples confirms colonization with probiotic yeast cells.
  • Bioanalytical analysis Detection of the altered bile salt composition confirms in situ production and active presence of API.
  • ELISA analysis of toxin A and/or B Levels of toxins A and/or B in cecum reflect progression of disease, and are lower in the animals treated with Sb BSH , or even lower in Sb BSH + drug treatment.
  • Histopathology Histopathological analysis shows more severe mucosal damage and inflammation induced by the C. difficile toxins in the controls versus the Sb BSH yeast strain.
  • Clostridioides difficile Sequence Type 11 a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance. mBio March/April 2019 Volume 10 Issue 2 e00446-19. doi.org/10.1128/mBio.00446-19. Lawley et al.; Antibiotic Treatment of Clostridium difficile Carrier Mice Triggers a Supershedder State, Spore-Mediated Transmission, and Severe Disease in Immunocompromised Hosts; Infection and Immunity; 2009; Vol.77, No.9; pp.3661–3669 Liong, M. T., & Shah, N. P. (2005).
  • Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains International Dairy Journal, 15(4), 391–398. https://doi.org/10.1016/J.IDAIRYJ.2004.08.007 Lupp et al.; Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae; Cell Host & Microbe; 2007; vol 2; pp.119–129. Marques, M. R. C., Loebenberg, R., & Almukainzi, M. (n.d.). Simulated Biological Fluids with Possible Application in Dissolution Testing.
  • Saccharomyces boulardii CNCM I-745 Improves Intestinal Enzyme Function: A Trophic Effects Review. Clinical Medicine Insights: Gastroenterology, 11, 117955221775267. https://doi.org/10.1177/1179552217752679 Moré, Margret Irmgard, & Swidsinski, A. (2015). Saccharomyces boulardii CNCM I-745 supports regeneration of the intestinal microbiota after diarrheic dysbiosis – a review. Clinical and Experimental Gastroenterology, 8, 237. https://doi.org/10.2147/CEG.S85574 Nielsen, H. (2019). SignalP 5.0 improves signal peptide predictions using deep neural networks.
  • a delivery vehicle comprising a genetically modified S. boulardii host cell comprising one or more heterologous polynucleotides encoding and producing a heterologous enzyme API capable of converting a compound which is inactive in the treatment of one or more diseases in a mammal into a compound which is active in the treatment of the one or more diseases in said mammal, wherein the vehicle is suitable for administering to the mammal and wherein the host cell is capable of producing and delivering the produced enzyme API in situ of the location in the mammal body in need of preventing, treating and/or relieving the disease.
  • Item 2. The delivery vehicle of item 1, wherein the API is an organic molecule having a molecular weight of more than 200 g/mol.
  • Item 3. The delivery vehicle of any preceding item, wherein the API is a polypeptide.
  • Item 4. The delivery vehicle of any preceding item, wherein the API is an enzyme.
  • Item 5. The delivery vehicle of any preceding item, wherein the API is capable of in situ converting a compound which is inactive in the prevention, treatment and/or relief of one or more diseases into a compound which is active in the prevention, treatment and/or relief of one or more diseases.
  • Item 6. The delivery vehicle of any preceding item comprising one or more polynucleotides encoding the API.
  • the delivery vehicle of any preceding item comprising a microbial host cell comprising the one or more polynucleotides encoding the API.
  • Item 8 The delivery vehicle of item 7, wherein vehicle is the microbial host cell.
  • Item 9. The delivery vehicle of item 8, wherein the microbial host cell is genetically modified.
  • Item 10. The delivery vehicle of items 7 to 9, wherein the one or more polynucleotides encoding the API is heterologous to the genetically modified cell.
  • BSH Bile Salt Hydrolase
  • GCDCA glycochenodeoxycholic acid
  • CA glycocholic acid
  • DCA deoxycholic acid
  • CDCA chenodeoxycholic acid
  • the API is a BSH comprising a polypeptide selected from: a) a polypeptide which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the mature BSH enzyme of anyone of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 111; b) a polypeptide encoded by a polynu
  • Item 15 The delivery vehicle of any preceding item, wherein the API is a BSH comprising a polypeptide selected from: a) a polypeptide which is at least 90% identical to the mature BSH enzyme of SEQ ID NO: 1; or b) a polypeptide encoded by a polynucleotide which is at least 90% identical to the polynucleotide comprised in SEQ ID NO: 2 encoding the mature polypeptide of SEQ ID NO: 1.
  • Item 16 The delivery vehicle of any preceding item comprising a microbial host cell, wherein the cell is a fungus or a bacterium.
  • the delivery vehicle of item 16 wherein the fungus is selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia.
  • the yeast is selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces.
  • the delivery vehicle of item 18, wherein the yeast host cell is selected from the species consisting of Kluyveromyces lactis, Saccharomyces boulardii, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Zygosaccharomyces spp., Schizosaccharomyces pombe, and Yarrowia lipolytica.
  • the yeast host cell is Saccharomyces boulardii. Item 21.
  • the delivery vehicle of item 16 wherein the bacterium is selected from the genera consisting of Lactobacillus, Leuconostoc, streptomyces, Pediococcus, Lactococcus, Bifidobacterium, Weissella, Streptococcus, Komagataeibacter, Acetobacter, and Gluconacetobacter. Item 22.
  • the delivery vehicle of item 21, wherein the bacterium host cell is selected from the species consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bacteroides ovatus, Bacteroides fragilis, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus gallinarum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paraplantarum, Lactobacillus coryniformis, Lactobacillus pentosus, and Lactobacillus fermentum, Lactobacillus delbrueckii subsp.
  • the bacterium host cell is selected from the species consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bacteroides ovatus, Bacteroides fragilis, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus gallinarum, Lactobacillus reuter
  • the delivery vehicle of item 20 wherein the vehicle is a genetically modified yeast cell of the species S. boulardii comprising and expressing a heterologous gene encoding a BSH enzyme and thereby producing said BSH enzyme.
  • Item 24 The delivery vehicle of any preceding item comprising a microbial host cell, wherein the cell further comprises at least one transporter molecule facilitating secretion of the API.
  • Item 25 The delivery vehicle of any preceding item comprising a microbial host cell, wherein one or more native genes of the cell is overexpressed, attenuated, disrupted and/or deleted.
  • the delivery vehicle of any preceding item comprising a genetically modified Saccharomyces boulardii modified by overexpressing one or more native genes KEX2, BIP, PDI, HAC1, SSO2, ERO1, COG5, and/or a functional deletion or downregulation of hda2, vps5 and tda3.
  • Item 27 The delivery vehicle of any preceding item comprising a microbial host cell, wherein the cell comprises at least 2 copies of a polynucleotide encoding the API.
  • Item 28. The delivery vehicle of any preceding item, wherein the vehicle is coated by a protective coating.
  • the delivery vehicle of any preceding item wherein the vehicle is encapsulated by a membrane, in a capsule, microcapsule, sphere and/or microsphere.
  • Item 30 The delivery vehicle of items 28 to 29, wherein the coating, membrane, capsule, microcapsule, sphere and/or microsphere is enteric.
  • Item 31 The delivery vehicle of items 28 to 30, wherein the enteric coating or membrane is triggered to release the vehicle and/or the API by pH, by osmotic pressure, by enzymatic digestion and/or by time-release.
  • Item 32 The delivery vehicle of any preceding item, wherein the vehicle is encapsulated by a membrane, in a capsule, microcapsule, sphere and/or microsphere.
  • the delivery vehicle of items 28 to 31, wherein the coating, capsule, microcapsule, sphere and/or microsphere comprise one or more materials selected from gums, proteins, waxes, polyols, alginates, starches, dextrans and chitosans.
  • Item 36 The delivery vehicle of items 28 to 35, wherein the vehicle comprise a microbial cell and the coating or membrane is impermeable to the cell.
  • Item 37 The delivery vehicle of items 28 to 36, wherein the coating or membrane is made of alginate- poly lysine-alginate (APA).
  • Item 38 The delivery vehicle of item 37, wherein the coating or membrane is made of materials selected from Alginate/Poly-L-lysine/Alginate (APA), Alginate/Poly-L-lysine/Pectin/Poly-L- lysine/Alginate (APPPA), Alginate/Poly-L-lysine/Pectin/Poly-L-lysine/Pectin (APPPP), and Alginate/Poly-L-lysine/Chitosan/Poly-L-lysine/Alginate (APCPA).
  • APA Alginate/Poly-L-lysine/Alginate
  • APPPA Alginate/Poly-L-lysine/Pectin/Poly-L-lysine/Pectin
  • Item 39 The delivery vehicle of any preceding item, wherein the vehicle is a genetically modified S. boulardii host cell comprising one or more heterologous polynucleotides encoding and producing a heterologous enzyme API capable of converting a compound which is inactive in the treatment of one or more diseases in a mammal into a compound which is active in the treatment of the one or more diseases in said mammal, wherein the vehicle is suitable for administering to the mammal and wherein the host cell is capable of producing and delivering the produced enzyme API in situ of the location in the mammal body in need of preventing, treating and/or relieving the disease.
  • the vehicle is suitable for administering to the mammal and wherein the host cell is capable of producing and delivering the produced enzyme API in situ of the location in the mammal body in need of preventing, treating and/or relieving the disease.
  • a polynucleotide construct comprising a polynucleotide sequence encoding an API of any preceding item, operably linked to one or more control sequences.
  • Item 41 The polynucleotide construct of item 40, wherein the control sequence is heterologous to the polynucleotide.
  • Item 42 The polynucleotide construct of item 41, wherein the construct is an expression vector.
  • Item 43 The vehicle of any preceding item comprising a microbial host cell comprising the polynucleotide construct of items 40 to 42.
  • Item 44. A cell culture, comprising the microbial host cell of any preceding item and a growth medium.
  • a method for producing the cell culture of item 43 comprising a) culturing the microbial host cell of any preceding item at conditions allowing growth of the microbial host cell; and b) optionally recovering and/or isolating the cell culture.
  • Item 46 A method for producing the cell culture of item 43 comprising a) culturing the microbial host cell of any preceding item at conditions allowing growth of the microbial host cell; and b) optionally recovering and/or isolating the cell culture.
  • the method of items 45 further comprising one or more elements selected from: a) culturing the cell culture in a nutrient medium; b) culturing the cell culture under aerobic or anaerobic conditions; c) culturing the cell culture under agitation; d) culturing the cell culture at a temperature of between 25 °C to 50 °C; e) culturing the cell culture at a pH of between 3-9; and f) culturing the cell culture for between 10 hours to 30 days. and having a cell density of at least 1-3x10 9 CFU/ml.
  • Item 47 A fermentation composition comprising the cell culture of item 44.
  • the fermentation composition of item 47 comprising the API and one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and/or amino acids of the fermentation; wherein the concentration of the API is at least 1 mg/l composition.
  • Item 49 The fermentation composition of items 47 to 48, having a cell density of at least 10 7 CFU/ml.
  • Item 50. A composition comprising the vehicle, and/or cell culture of any preceding item and one or more carriers, agents, additives and/or excipients.
  • a pharmaceutical composition comprising the vehicle, and/or cell culture of any preceding item and one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • Item 52 A pharmaceutical composition comprising the vehicle, and/or cell culture of any preceding item and one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • the pharmaceutical composition of item 51 wherein the pharmaceutical composition is in form of a powder, a tablet or a capsule.
  • Item 53 The pharmaceutical composition of item 51, wherein the pharmaceutical composition is in form of a pharmaceutical solution, suspension, lotion or ointment.
  • Item 54 The pharmaceutical composition of items 51 to 53 for use as a medicament for prevention, treatment and/or relief of a disease in a mammal.
  • Item 55 The pharmaceutical composition of item 54 for use in the prevention, treatment and/or relief of CDI and/or CDI induced colitis.
  • Item 56 The pharmaceutical composition of item 51, wherein the pharmaceutical composition is in form of a powder, a tablet or a capsule.
  • the pharmaceutical composition of item 55 wherein the API is BSH and the treatment comprises contacting a pathogenic strain of the genus Clostridioides in the presence of a conjugated bile acid with the pharmaceutical composition at conditions allowing the vehicle and/or cell culture to produce and deliver BSH in amounts converting the conjugated bile acid into therapeutically effective amounts of deconjugated bile acids inhibiting germination and/or proliferation of the pathogenic strain.
  • Item 57 The pharmaceutical composition of item 56, wherein the prevention, treatment and/or relief is performed in situ of the location of the pathogenic strain in and/or on the body of the mammal.
  • Item 58 The pharmaceutical composition of item 55, wherein the API is BSH and the treatment comprises contacting a pathogenic strain of the genus Clostridioides in the presence of a conjugated bile acid with the pharmaceutical composition at conditions allowing the vehicle and/or cell culture to produce and deliver BSH in amounts converting the conjugated bile acid into therapeutically effective amounts of deconjugated
  • the pharmaceutical composition of items 54 to 57 wherein the composition is administered daily in an amount of at least 10 mg or at least 10 6 CFU per kg body mass of the mammal to be treated.
  • Item 59 The pharmaceutical composition of items 54 to 58, wherein the composition is administered parenterally.
  • Item 60 The pharmaceutical composition of item 59, wherein the parenteral administration is ocular administration.
  • Item 61 The pharmaceutical composition of item 59, wherein the parenteral administration is pulmonary administration.
  • Item 63 The pharmaceutical composition of items 54 to 58, wherein the composition is administered topically.
  • Item 64 The pharmaceutical composition of items 54 to 58, wherein the composition is administered daily in an amount of at least 10 mg or at least 10 6 CFU per kg body mass of the mammal to be treated.
  • Item 60 The pharmaceutical composition of items 54 to 58, wherein the composition is administered parenterally.
  • Item 60 The
  • a method for preparing the pharmaceutical composition of item 51 to 63 comprising mixing the vehicle, and/or the cell culture of any preceding item with one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • Item 65 A method for preventing, treating and/or relieving a disease comprising administering the pharmaceutical composition of items 51 to 53 to a mammal in an amount for the vehicle and/or cell culture to produce and deliver in situ a therapeutically effective amount of the API.
  • Item 66. The method of item 65, wherein the disease is CDI and/or CDI induced colitis.
  • the method of item 66 wherein the prevention, treatment and/or relief is inhibiting germination or proliferation of a pathogenic strain of the genus Clostridioides, the API is BSH and the prevention, treatment and/or relief comprises contacting the pathogenic strain in the presence of a conjugated bile acid with the pharmaceutical composition at conditions allowing the vehicle and/or cell culture to produce and deliver BSH in amounts converting the conjugated bile acid into therapeutically effective amounts of deconjugated bile acids inhibiting germination and/or proliferation of the pathogenic strain.
  • the method is performed in situ of the pathogenic strain in and/or on the body of a mammal.
  • a delivery vehicle comprising a genetically modified microbial host cell comprising one or more heterologous polynucleotides encoding and producing a one or more enzyme active pharmaceutical ingredient (API), wherein the vehicle is suitable for administering to the mammal and wherein the modified microbial host cell is capable of producing and delivering the one or more enzyme API in situ of the location in the body of a mammal in need of preventing, treating and/or relieving a disease.
  • Item 76 The delivery vehicle of item 75, wherein at least one of the one or more heterologous polynucleotides of the genetically modified microbial host cell encodes a heterologous enzyme API or overexpresses a native enzyme API compared to an unmodified microbial host cell.
  • Item 77 The delivery vehicle of item 75 or 76, wherein the one or more enzyme API is capable of enzymatic activity in conditions found within the mammalian gastrointestinal tract.
  • Item 78. The delivery vehicle of any of items 75 to 77, wherein the API is capable of in situ converting a compound which is inactive in the prevention, treatment and/or relief of one or more diseases into a compound which is active in the prevention, treatment and/or relief of one or more diseases.
  • the delivery vehicle of any of items 75 to 78, wherein the one or more diseases is a Clostridioides infection, Clostridioides infection induced colitis, obesity, type 2 diabetes, cardiovascular disease, colon cancer, polycystic ovary syndrome, a neurological disease, diseases of the liver including nonalcoholic steatohepatitis, cirrhosis and/or liver cancer.
  • the one or more diseases is a Clostridioides infection, Clostridioides infection induced colitis, obesity, type 2 diabetes, cardiovascular disease, colon cancer, polycystic ovary syndrome, a neurological disease, diseases of the liver including nonalcoholic steatohepatitis, cirrhosis and/or liver cancer.
  • Item 80 The delivery vehicle of item 79, wherein the Clostridioides species is C. difficile.
  • BSH Bile Salt Hydrolase
  • the delivery vehicle of item 81 wherein the conjugated bile salt is selected from glycocholic acid (GCA); taurocholic acid (TCA); glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA); taurochenodeoxycholic acid (TCDCA); and glycochenodeoxycholic acid (GCDCA) and the deconjugated bile salt is selected from cholic acid (CA); deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) respectively.
  • GCDCA glycocholic acid
  • CA glycodeoxycholic acid
  • DCA deoxycholic acid
  • CDCA chenodeoxycholic acid
  • the API is a BSH comprising a polypeptide selected from: a) a polypeptide which is at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the mature BSH enzyme of anyone of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, or 111; b) a polypeptide encoded by a polypeptide encoded by a polypeptide encoded
  • Item 84 The delivery vehicle of any of items 75 to 83, wherein the heterologous enzyme API is a BSH comprising a polypeptide selected from: a) a polypeptide which is at least 90% identical to the mature BSH enzyme of SEQ ID NO: 1; or b) a polypeptide encoded by a polynucleotide which is at least 90% identical to the polynucleotide comprised in SEQ ID NO: 2 encoding the mature polypeptide of SEQ ID NO: 1.
  • Item 85 The delivery vehicle of any of items 75 to 84 comprising a microbial host cell, wherein the cell is a fungus or a bacterium.
  • Item 86 The delivery vehicle of any of items 75 to 84 comprising a microbial host cell, wherein the cell is a fungus or a bacterium.
  • the delivery vehicle of item 85 wherein the fungus is selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Zygomycota, Oomycota and Microsporidia.
  • the delivery vehicle of item 85 or 86, wherein the fungus is a yeast is selected from the genera consisting of Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, and Schizosaccharomyces.
  • Item 88 The delivery vehicle of item 85, wherein the fungus is selected from the phylas consisting of Ascomycota, Basidiomycota, Neocallimastigomycota, Glomeromycota, Blastocladiomycota, Chytridiomycota, Z
  • the delivery vehicle of item 87 wherein the yeast host cell is selected from the species consisting of Kluyveromyces lactis, Saccharomyces boulardii, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Zygosaccharomyces spp., Schizosaccharomyces pombe, and Yarrowia lipolytica.
  • Item 89 The delivery vehicle of item 88, wherein the yeast host cell is Saccharomyces boulardii. Item 90.
  • the delivery vehicle of item 85 wherein the bacterium is selected from the genera consisting of Lactobacillus, Leuconostoc, Streptomyces, Pediococcus, Lactococcus, Bifidobacterium, Weissella, Streptococcus, Komagataeibacter, Acetobacter, and Gluconacetobacter. Item 91.
  • the delivery vehicle of item 90 wherein the bacterium host cell is selected from the species consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bacteroides ovatus, Bacteroides fragilis, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus gallinarum, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus paraplantarum, Lactobacillus coryniformis, Lactobacillus pentosus, and Lactobacillus fermentum, Lactobacillus delbrueckii subsp.
  • the bacterium host cell is selected from the species consisting of Lactobacillus acidophilus, Lactobacillus bulgaricus, Bacteroides ovatus, Bacteroides fragilis, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus gallinarum, Lactobacillus reuter
  • the delivery vehicle of any of items 75 to 91 comprising a microbial host cell, wherein the cell further comprises at least one transporter molecule facilitating secretion of an API of any of the preceding items.
  • Item 93 The delivery vehicle of any of items 75 to 92 comprising a microbial host cell, wherein one or more native genes of the microbial host cell is overexpressed, attenuated, disrupted and/or deleted.
  • the delivery vehicle of item 89 wherein the microbial host comprises a genetically modified Saccharomyces boulardii modified by overexpressing one or more native genes KEX2, BIP, PDI, HAC1, SSO2, ERO1, COG5, and/or a functional deletion or downregulation of had2, vps5 and tda3 and the the API is a BSH.
  • Item 95 The delivery vehicle of any of items 75 to 94 comprising a microbial host cell, wherein the cell comprises at least 2 copies of a polynucleotide encoding an API of any of the preceding items.
  • Item 96 The delivery vehicle of any of items 75 to 95, wherein the vehicle is coated by a protective coating.
  • Item 97 The delivery vehicle of any of items 75 to 95, wherein the vehicle is coated by a protective coating.
  • Item 98. The delivery vehicle of items 96 to 97, wherein the coating, membrane, capsule, microcapsule, sphere and/or microsphere is enteric.
  • Item 99. The delivery vehicle of items 96 to 98, wherein the enteric coating or membrane is triggered to release the vehicle and/or the API by pH, by osmotic pressure, by enzymatic digestion and/or by time-release.
  • Item 100 The delivery vehicle of any of items 75 to 96, wherein the vehicle is encapsulated by a membrane, in a capsule, microcapsule, sphere and/or microsphere.
  • APA Alginate/Poly-L-lysine/Alginate
  • APPPA Alginate/Poly-L-lysine/Pectin/Poly-L-lysine/Alginate
  • APPPP Alginate/Poly-L-lysine/Chitosan/Poly-L-lysine/Alginate
  • APIPA Al
  • a polynucleotide construct comprising a polynucleotide sequence encoding an API of any preceding item, operably linked to one or more control sequences.
  • Item 106. The polynucleotide construct of item 105, wherein the control sequence is heterologous to the polynucleotide.
  • Item 107. The polynucleotide construct of item 106, wherein the construct is an expression vector.
  • Item 108. The vehicle of any preceding item comprising a microbial host cell comprising the polynucleotide construct of items 106 to 107.
  • Item 109. A cell culture, comprising the microbial host cell of any preceding item and a growth medium or fermentation medium.
  • the cell culture of item 109 comprising the one or more API and one or more compounds selected from trace metals, vitamins, salts, yeast nitrogen base, carbon source, YNB, and/or amino acids of the fermentation; wherein the concentration of the one or more API is at least 1 mg/l composition.
  • Item 111. The cell culture of items 109 to 110, having a cell density of at least 10 7 CFU/ml.
  • Item 112. A method for producing the cell culture of any of items 109 to 111, comprising a) culturing the microbial host cell of any preceding item at conditions allowing growth of the microbial host cell; and b) optionally recovering and/or isolating the cell culture.
  • Item 113 A method for producing the cell culture of any of items 109 to 111, comprising a) culturing the microbial host cell of any preceding item at conditions allowing growth of the microbial host cell; and b) optionally recovering and/or isolating the cell culture.
  • a composition comprising the delivery vehicle, and/or cell culture of any preceding item and one or more carriers, agents, additives and/or excipients.
  • Item 115. A pharmaceutical composition comprising the delivery vehicle, and/or cell culture of any preceding item and one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • Item 116. The pharmaceutical composition of item 115, wherein the pharmaceutical composition is in form of a powder, a tablet or a capsule.
  • Item 117. The pharmaceutical composition of item 115, wherein the pharmaceutical composition is in form of a pharmaceutical solution, suspension, lotion or ointment.
  • Item 118. The pharmaceutical composition of items 115 to 117 for use as a medicament for prevention, treatment and/or relief of a disease in a mammal.
  • Item 119 The pharmaceutical composition of item 118 for use in the prevention, treatment and/or relief of CDI and/or CDI induced colitis, obesity, type 2 diabetes, cardiovascular disease, colon cancer, polycystic ovary syndrome, a neurological disease, diseases of the liver including nonalcoholic steatohepatitis, cirrhosis and/or liver cancer.
  • Item 120 The pharmaceutical composition of item 118 for use in the prevention, treatment and/or relief of CDI and/or CDI induced colitis, obesity, type 2 diabetes, cardiovascular disease, colon cancer, polycystic ovary syndrome, a neurological disease, diseases of the liver including nonalcoholic steatohepatitis, cirrhosis and/or liver cancer.
  • the pharmaceutical composition of item 119 wherein the API is a BSH, and the treatment comprises contacting a pathogenic strain of the genus Clostridioides in the presence of a conjugated bile acid with the pharmaceutical composition at conditions allowing the vehicle and/or cell culture to produce and deliver BSH in situ of the location of the pathogenic strain in amounts converting the conjugated bile acid into therapeutically effective amounts of deconjugated bile acids inhibiting germination and/or proliferation of the pathogenic strain.
  • Item 121 The pharmaceutical composition of any of items 115 to 120, wherein the composition is administered daily in an amount of at least 10 mg or at least 10 6 CFU per kg body mass of the mammal to be treated.
  • Item 122 The pharmaceutical composition of any of items 115 to 120, wherein the composition is administered daily in an amount of at least 10 mg or at least 10 6 CFU per kg body mass of the mammal to be treated.
  • Item 123. A method for preparing the pharmaceutical composition of item 115 to 122 comprising mixing the vehicle, and/or the cell culture of any preceding item with one or more pharmaceutical grade excipient, additives and/or adjuvants.
  • Item 124. A method for preventing, treating and/or relieving a disease comprising administering the pharmaceutical composition of items 115 to 123 to a mammal in an amount for the vehicle and/or cell culture to produce and deliver in situ a therapeutically effective amount of the one or more API.
  • the method of item 125 or 126 wherein the method is performed in situ of the Clostridioides infection in and/or on the body of a mammal.
  • Item 128 The method of any of items 124 to 127, wherein the composition is administered daily in an amount of at least 10 mg or and least 10 6 CFU per kg body mass of the mammal to be treated.
  • Item 129 The method of any of items 124 to 128, wherein the composition is administered enterally, orally, topically or rectally.

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Abstract

La présente invention concerne des véhicules d'administration comprenant un système produisant un ou plusieurs ingrédients pharmaceutiques actifs (API) pouvant prévenir, traiter et/ou soulager une ou plusieurs maladies chez un mammifère, le véhicule comprenant des cellules hôtes microbiennes génétiquement modifiées appropriées pour une administration au mammifère, le véhicule permettant d'administrer l'API produit in situ à un emplacement donné dans le corps du mammifère pour lequel une prévention, un traitement et/ou une atténuation de la maladie est nécessaire.
PCT/EP2020/075152 2019-09-09 2020-09-09 Véhicule de distribution pour l'administration in situ d'agents pharmaceutiques WO2021048172A2 (fr)

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Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000787A1 (fr) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Systeme d'expression de fusarium non pathogene, non toxicogene, non toxique, et promoteurs et terminateurs utilises dans ce systeme
WO2000024883A1 (fr) 1998-10-26 2000-05-04 Novozymes A/S Etablissement et criblage d'une banque d'adn d'interet dans des cellules fongiques filamenteuses
WO2000056900A2 (fr) 1999-03-22 2000-09-28 Novo Nordisk Biotech, Inc. Promoteurs exprimant les genes d'une cellule fongique
WO2005100573A2 (fr) 2004-04-16 2005-10-27 Dsm Ip Assets B.V. Promoteurs fongiques destines a l'expression d'un gene dans une cellule fongique
WO2006092396A1 (fr) 2005-03-01 2006-09-08 Dsm Ip Assets B.V. Promoteurs d'aspergillus utiles pour l'expression d'un gene dans une cellule fongique
WO2008098933A1 (fr) 2007-02-15 2008-08-21 Dsm Ip Assets B.V. Cellule hôte recombinante pour la production d'un composé d'intérêt
WO2010124387A1 (fr) 2009-05-01 2010-11-04 Micropharma Limited Compositions bactériennes destinées au traitement et à la prophylaxie de maladies dégénératives
WO2015168534A1 (fr) 2014-05-02 2015-11-05 Novogy, Inc. Traitement thérapeutique de déséquilibres microbiens gastro-intestinaux par déplacement de microbes compétitif
CN106399284A (zh) 2016-12-14 2017-02-15 吴银娣 一种两阶段发酵提高毕赤酵母产胆盐水解酶的方法
CN106591272A (zh) 2016-12-14 2017-04-26 曹书华 一种酶活提高的胆盐水解酶突变体
CN106754844A (zh) 2016-12-14 2017-05-31 曹书华 一种提高胆盐水解酶分泌效率的方法
CN106754445A (zh) 2016-12-14 2017-05-31 曹书华 一种产胆盐水解酶的重组毕赤酵母
CN106754843A (zh) 2016-12-14 2017-05-31 曹书华 一种提高重组大肠杆菌胆盐水解酶产量的方法
WO2017123592A1 (fr) 2016-01-11 2017-07-20 Synlogic, Inc. Bactérie manipulée pour traiter des troubles associés aux sels biliaires
US20170360850A1 (en) 2014-12-22 2017-12-21 Massachusetts Institute Of Technology Probiotic organisms for diagnosis, monitoring, and treatment of inflammatory bowel disease
WO2019053218A1 (fr) 2017-09-15 2019-03-21 Vib Vzw Moyens et procédés permettant de moduler la puissance probiotique de la levure saccharomyces boulardii

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3011966A1 (fr) * 2014-08-08 2016-04-27 Institut National De La Recherche Agronomique Compositions pour l'inhibition de Giardia Lamblia
WO2016210384A2 (fr) * 2015-06-25 2016-12-29 Synlogic, Inc. Bactéries manipulées pour traiter des maladies métaboliques
EP3402497A1 (fr) * 2016-01-11 2018-11-21 Synlogic Operating Company, Inc. Bactéries modifiées pour traiter des maladies métaboliques

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000787A1 (fr) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Systeme d'expression de fusarium non pathogene, non toxicogene, non toxique, et promoteurs et terminateurs utilises dans ce systeme
WO2000024883A1 (fr) 1998-10-26 2000-05-04 Novozymes A/S Etablissement et criblage d'une banque d'adn d'interet dans des cellules fongiques filamenteuses
WO2000056900A2 (fr) 1999-03-22 2000-09-28 Novo Nordisk Biotech, Inc. Promoteurs exprimant les genes d'une cellule fongique
WO2005100573A2 (fr) 2004-04-16 2005-10-27 Dsm Ip Assets B.V. Promoteurs fongiques destines a l'expression d'un gene dans une cellule fongique
WO2006092396A1 (fr) 2005-03-01 2006-09-08 Dsm Ip Assets B.V. Promoteurs d'aspergillus utiles pour l'expression d'un gene dans une cellule fongique
WO2008098933A1 (fr) 2007-02-15 2008-08-21 Dsm Ip Assets B.V. Cellule hôte recombinante pour la production d'un composé d'intérêt
WO2010124387A1 (fr) 2009-05-01 2010-11-04 Micropharma Limited Compositions bactériennes destinées au traitement et à la prophylaxie de maladies dégénératives
WO2015168534A1 (fr) 2014-05-02 2015-11-05 Novogy, Inc. Traitement thérapeutique de déséquilibres microbiens gastro-intestinaux par déplacement de microbes compétitif
US20170360850A1 (en) 2014-12-22 2017-12-21 Massachusetts Institute Of Technology Probiotic organisms for diagnosis, monitoring, and treatment of inflammatory bowel disease
WO2017123592A1 (fr) 2016-01-11 2017-07-20 Synlogic, Inc. Bactérie manipulée pour traiter des troubles associés aux sels biliaires
CN106399284A (zh) 2016-12-14 2017-02-15 吴银娣 一种两阶段发酵提高毕赤酵母产胆盐水解酶的方法
CN106754445A (zh) 2016-12-14 2017-05-31 曹书华 一种产胆盐水解酶的重组毕赤酵母
CN106754843A (zh) 2016-12-14 2017-05-31 曹书华 一种提高重组大肠杆菌胆盐水解酶产量的方法
CN106754844A (zh) 2016-12-14 2017-05-31 曹书华 一种提高胆盐水解酶分泌效率的方法
CN106591272A (zh) 2016-12-14 2017-04-26 曹书华 一种酶活提高的胆盐水解酶突变体
WO2019053218A1 (fr) 2017-09-15 2019-03-21 Vib Vzw Moyens et procédés permettant de moduler la puissance probiotique de la levure saccharomyces boulardii

Non-Patent Citations (79)

* Cited by examiner, † Cited by third party
Title
"RStudio: Integrated Development Environment for R", RSTUDIO TEAM, 2016, Retrieved from the Internet <URL:http://www.rstudio.com>
ALMAGRO ARMENTEROS, J. J.TSIRIGOS, K. D.SONDERBY, C. K.PETERSEN, T. N.WINTHER, 0.BRUNAK, S.NIELSEN, H.: "SignalP 5.0 improves signal peptide predictions using deep neural networks", NATURE BIOTECHNOLOGY, vol. 37, no. 4, 2019, pages 420 - 423, XP036900634, Retrieved from the Internet <URL:https://doi.org/10.1038/s41587-019-0036-z> DOI: 10.1038/s41587-019-0036-z
BAGHERPOUR, G.GHASEMI, H.ZAND, B.ZAREI, N.ROOHVAND, F.ARDAKANI, E. M.AZIZI, M.KHALAJ, V.: "Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice", FRONT. MICROBIOL., 13 April 2018 (2018-04-13), Retrieved from the Internet <URL:https://doi.org/10.3389/fmicb.2018.00723>
BARLOWE, C. K.MILLER, E. A.: "Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway", GENETICS, vol. 193, no. 2, 1 February 2013 (2013-02-01), pages 383 - 410, Retrieved from the Internet <URL:https://doi.org/10.1534/genetics.112.142810>
BEGLEY, M.HILL, C.GAHAN, C. G. M., BILE SALT HYDROLASE ACTIVITY IN PROBIOTICS, 2006
BEN THOMAS M ET AL., J. FOOD ENG., vol. 136, 2014, pages 1 - 8
BLEHAUT, H.MASSOT, J.ELMER, G. W.LEVY, R. H.: "Disposition kinetics of Saccharomyces boulardii in man and rat", BIOPHARMACEUTICS & DRUG DISPOSITION, vol. 10, no. 4, 1989, pages 353 - 364, Retrieved from the Internet <URL:https://doi.org/10.1002/bdd.2510100403>
BRANDVOLD, K. R.WEAVER, J. M.WHIDBEY, C.WRIGHT, A. T.: "A continuous fluorescence assay for simple quantification of bile salt hydrolase activity in the gut microbiome", SCIENTIFIC REPORTS, vol. 9, no. 1, 2019, pages 1359, Retrieved from the Internet <URL:https://doi.org/10.1038/s41598-018-37656-7>
BUCKLEY AMSPENCER JCANDLISH DIRVINE JJDOUCE GR: "Infection of hamsters with the UK Clostridium difficile ribotype 027 outbreak strain R20291", J MED MICROBIOL., vol. 60, 2011, pages 1174 - 1180
BUCKLEY, A. M.SPENCER, J.MACLELLAN, L. M.CANDLIS, D.IRVINE, J. J.DOUCE, G. R.: "Susceptibility of Hamsters to Clostridium difficile Isolates of Differing Toxinotype", PUBLIC LIBRARY OF SCIENCE, vol. 8, no. 5, 2013, pages e64121
BUFFIE: "Profound Alterations of Intestinal Microbiota following a Single Dose of Clindamycin Results in Sustained Susceptibility to Clostridium difficile-Induced Colitis", INFECTION AND IMMUNITY, vol. 80, no. 1, 2010, pages 62 - 73
CASTAGLIUOLO, I.RIEGLER, M. F.VALENICK, L.LAMONT, J. T.POTHOULAKIS, C.: "Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa", INFECT IMMUN, vol. 67, no. 1, 1999, pages 302 - 7
CHEN ET AL.: "A Mouse Model of Clostridium difficile - Associated Disease", GASTROENTEROLOGY, vol. 135, no. 6, 2008, pages 1984 - 1992, XP026147662, DOI: 10.1053/j.gastro.2008.09.002
CHEN, L. A.VAN MEERBEKE, S.ALBESIANO, E.GOODWIN, A.WU, S.YU, H.SEARS, C.: "Fecal detection of enterotoxigenic Bacteroides fragilis", EUROPEAN JOURNAL OF CLINICAL MICROBIOLOGY & INFECTIOUS DISEASES: OFFICIAL PUBLICATION OF THE EUROPEAN SOCIETY OF CLINICAL MICROBIOLOGY, vol. 34, no. 9, 2015, pages 1871 - 1877, Retrieved from the Internet <URL:https://doi.org/1O.1007/s10096-015-2425-7>
CHRISTIAENS, H.LEER, R. J.POUWELS, P. H.VERSTRAETE, W.: "Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 58, no. 12, 1992, pages 3792 - 3798, XP008038416, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pubmed/1476424>
CORZO, G.GILLILAND, S. E.: "Bile Salt Hydrolase Activity of Three Strains of Lactobacillus acidophilus", JOURNAL OF DAIRY SCIENCE, vol. 82, no. 3, 1999, pages 472 - 480, XP026993466, Retrieved from the Internet <URL:https://doi.org/10.3168/jds.S0022-0302(99)75256-2>
CULLEN ET AL., NUCLEIC ACIDS RES., vol. 15, 1987, pages 9163 - 9175
EUR. J. BIO-CHEM., vol. 223, 1994, pages 1 - 5
EUR. J. BIOCHEM., vol. 232, 1995, pages 1 - 6
EUR. J. BIOCHEM., vol. 237, 1996, pages 1 - 5
EUR. J. BIOCHEM., vol. 250, 1997, pages 1 - 6
EUR. J. BIOCHEM., vol. 264, 1999, pages 610 - 650
FATIMA, R.AZIZ, M.: "The Hypervirulent Strain of Clostridium Difficile: NAP1/B1/027 - A Brief Overview", CUREUS, vol. 11, no. 1, 2019, pages e3977, Retrieved from the Internet <URL:https://doi.org/10.7759/cureus.3977>
FRANZ, C. M. A. P.SPECHT, I.HABERER, P.HOLZAPFEL, W. H.: "Bile Salt Hydrolase Activity of Enterococci Isolated from Food: Screening and Quantitative Determination", JOURNAL OF FOOD PROTECTION, vol. 64, 2001, Retrieved from the Internet <URL:https://jfoodprotection.org/doi/pdfplus/10.4315/0362-028X-64.5.725>
GEMS ET AL., GENE, vol. 98, 1991, pages 61 - 67
GIEL, J. L.SORG, J. A.SONENSHEIN, A. L.ZHU, J.: "Metabolism of Bile Salts in Mice Influences Spore Germination", C. DIFFICILE. PLOS ONE, vol. 5, no. 1, 2010, pages 8740, Retrieved from the Internet <URL:https://doi.org/10.1371/journal.pone.0008740>
GOULDING, D.THOMPSON, H.EMERSON, J.FAIRWEATHER, N. F.DOUGAN, G.DOUCE, G. R.: "Distinctive Profiles of Infection and Pathology in Hamsters Infected with Clostridium difficile Strains 630 and B1", INFECT IMMUN., vol. 77, no. 12, 2009, pages 5478 - 5485
GUOSHERMAN, MOL. CELLULAR BIOL., vol. 15, 1995, pages 5983 - 5990
H. WEN ET AL., AAPS J., vol. 17, no. 6, November 2015 (2015-11-01), pages 1327 - 1340
HANSEN EH: "Faculty of Pharmaceutical Science", 2009, UNIVERSITY OF COPENHAGEN, article "Enzymatic glycosylation of small molecule drugs: A strategy to improve absorption and distribution [Ph.D thesis"
HAY, D. W.CAREY, M. C.: "Chemical species of lipids in bile", HEPATOLOGY (BALTIMORE, MD., vol. 12, no. 3, 1990, pages 6S - 14S
HEIDEBRECHT, H. J.WEISS, W. J.PULSE, M.LANGE, A.GISCH, K.KLIEM, H.VON EICHEL-STREIBER, C.: "Treatment and Prevention of Recurrent Clostridium difficile Infection with Functionalized Bovine Antibody-Enriched Whey in a Hamster Primary Infection Model", TOXINS, vol. 11, no. 2, 2019, pages 98
HONG HAHITRI KHOSSEINI SKOTOWICZ NBRYAN DMAWAS FWILKINSON AJVAN BROEKHOVEN AKEARSEY JCUTTING SM: "Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile", INFECT IMMUN, vol. 85, no. 4, 2017
HONG, H. A.FERREIRA, W. T.HOSSEINI, S.ANWAR, S.HITRI, K.WILKINSON, A. J.CUTTING, S. M: "The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile", THE JOURNAL OF INFECTIOUS DISEASES, vol. 216, no. 11, 2017, pages 1452 - 1459
HOU, J.TYO, K. E. J.LIU, Z.PETRANOVIC, D.NIELSEN, J.: "Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae", FEMS YEAST RESEARCH, vol. 12, no. 5, 2012, pages 491 - 510, Retrieved from the Internet <URL:https://doi.org/10.1111/j.1567-1364.2012.00810.x>
HUANG, M.WANG, G.QIN, J.PETRANOVIC, D.NIELSEN, J.: "Engineering the protein secretory pathway of Saccharomyces cerisiae improved protein production", PNAS, vol. 115, no. 47, 20 November 2018 (2018-11-20), pages E11025 - E11032
HUDSON, L. E.FASKEN, M. B.MCDERMOTT, C. D.MCBRIDE, S. M.KUIPER, E. G.GUILIANO, D. B.CORBETT, A. H.LAMB, T. J.: "Functional Heterologous Protein Expression by Genetially Engineered Probiotic Yeast Saccharomyces bouladii", PLOS ONE, vol. 9, no. 11, November 2014 (2014-11-01), pages e112660
ICHIHARA KFUKUBAYASHI Y: "Preparation of fatty acid methyl esters for gas-liquid chromatography", J LIPID RES, vol. 51, 2010, pages 635 - 40
J. KIM, JOURNAL OF PHARMACEUTICAL INVESTIGATION ONLINE, ISSN: 2093-6214
J. LID.J. MOONEY, NAT. REV. MATER., vol. 1, no. 12, December 2016 (2016-12-01), pages 16071
JENSEN, N. B.STRUCKO, T.KILDEGAARD, K. R.DAVID, F.MAURY, J.MORTENSEN, U. H.BORODINA, I.: "EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae", FEMS YEAST RESEARCH, vol. 14, no. 2, 2014, pages 238 - 248, XP055273409, Retrieved from the Internet <URL:https://doi.org/10.1111/1567-1364.12118> DOI: 10.1111/1567-1364.12118
JIA, B.PARK, D.HAHN, Y.JEON, C. O.: "Metagenomic analysis of the human microbiome reveals the association between the abundance of gut bile salt hydrolases and host health", GUT MICROBES, vol. 11, no. 5, 2020, pages 1300 - 1313, Retrieved from the Internet <URL:https://doi.org/10.1080/19490976.2020.1748261>
JIANG, J.HANG, X.ZHANG, M.LIU, X.LI, D.YANG, H.: "Diversity of bile salt hydrolase activities in different lactobacilli toward human bile salts", ANNALS OF MICROBIOLOGY, vol. 60, no. 1, 2010, pages 81 - 88, Retrieved from the Internet <URL:https://doi.org/10.1007/sl3213-009-0004-9>
JOHN S. FORDTRAN, PROC (BAYL UNIV MED CENT, vol. 19, no. 1, January 2006 (2006-01-01), pages 3 - 12
KHATRI, I.TOMAR, R.GANESAN, K.PRASAD, G. S.SUBRAMANIAN, S.: "Complete genome sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii", SCIENTIFIC REPORTS, vol. 7, no. 1, 2017, pages 371, Retrieved from the Internet <URL:https://doi.org/10.1038/s41598-017-00414-2>
KJELDSEN, T.: "Yeast secretory expression of insulin precursors", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 54, no. 3, 2000, pages 277 - 286, XP008095326, Retrieved from the Internet <URL:https://doi.org/10.1007/s002530000402> DOI: 10.1007/s002530000402
KNIGHT, D. R.KULLIN, B.ANDROGA, G. O.BARBUT, F.ECKERT, K.JOHNSON, S.SPIGAGLIA, P.TATEDA, K.TSAI P. J.RILEY, T. V.: "Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance", MBIO, vol. 10, no. 2, 2019, pages e00446 - 19
L. LIU ET AL.: "pH- Responsive carriers for oral drug delivery: challenges and opportunities of current platforms", DRUG DELIVERY, vol. 24, no. 1, 2017
LAWLEY ET AL.: "Antibiotic Treatment of Clostridium difficile Carrier Mice Triggers a Supershedder State, Spore-Mediated Transmission, and Severe Disease in Immunocompromised Hosts", INFECTION AND IMMUNITY, vol. 77, no. 9, 2009, pages 3661 - 3669
LIONG, M. T.SHAH, N. P.: "Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains", INTERNATIONAL DAIRY JOURNAL, vol. 15, no. 4, 2005, pages 391 - 398, XP004715114, Retrieved from the Internet <URL:https://doi.org/10.1016/J.iDAiRYJ.2004.08.007> DOI: 10.1016/j.idairyj.2004.08.007
LUPP ET AL.: "Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae", CELL HOST & MICROBE, vol. 2, 2007, pages 119 - 129
M. GOVENDER, AAPS PHARMSCITECH., vol. 15, no. 1, February 2014 (2014-02-01), pages 29 - 43
MARQUES, M. R. C.LOEBENBERG, R.ALMUKAINZI, M. (N.D., SIMULATED BIOLOGICAL FLUIDS WITH POSSIBLE APPLICATION IN DISSOLUTION TESTING, Retrieved from the Internet <URL:https://doi.org/10.14227/DT180311P15>
MCDONALD, L. C.GERDING, D.N.JOHNSON, S.BAKKEN, J. S.CARROLL, K. C.COFFIN, S. E.DUBBERKE, E. R.GAREY, K. W.GOULD, C. V.KELLY, C.: "Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA", CLINICAL INFECTIOUS DISEASES, vol. 66, no. 7, 1 April 2018 (2018-04-01), pages e1 - e48
MIKKELSEN, M. D.BURON, L. D.SALOMONSEN, B.OLSEN, C. E.HANSEN, B. G.MORTENSEN, U. H.HALKIER, B. A.: "Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform", METABOLIC ENGINEERING, vol. 14, no. 2, 2012, pages 104 - 111, XP028466090, Retrieved from the Internet <URL:https://doi.Org/10.1016/j.ymben.2012.01.006> DOI: 10.1016/j.ymben.2012.01.006
MORE, MARGRET IRMGARDSWIDSINSKI, A: "Saccharomyces boulardii CNCM 1-745 supports regeneration of the intestinal microbiota after diarrheic dysbiosis – a review", CLINICAL AND EXPERIMENTAL GASTROENTEROLOGY, vol. 8, 2015, pages 237, Retrieved from the Internet <URL:https://doi.org/10.2147/CEG.S85574>
MORE, MARGRET IVANDENPLAS, Y.: "Saccharomyces boulardii CNCM 1-745 Improves Intestinal Enzyme Function: A Trophic Effects Review", CLINICAL MEDICINE INSIGHTS: GASTROENTEROLOGY, vol. 11, 2018, pages 117955221775267, Retrieved from the Internet <URL:https://doi.org/10.1177/1179552217752679>
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
PERMPOONPATTANA, P.HONG, H. A.PHETCHARABURANIN, J.: "Immunization with Bacillus spores expressing toxin A peptide repeats protects against infection with Clostridium difficile strains producing toxins A and B", INFECTION AND IMMUNITY, vol. 79, 2011, pages 2295 - 302, XP008160814, DOI: 10.1128/IAI.00130-11
PHETCHARABURANIN, J.HONG, H. A.COLENUTT, C.: "The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile", MOL MICROBIOL, vol. 92, 2014, pages 1025 - 38
POTHOULAKIS, C.THEOHARIDES, T. C.DICKEY, B. F.: "Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein", J CLIN INVEST., vol. 88, no. 1, 1991, pages 119 - 125
RAM, K.WICKHAM, H., WESANDERSON: A WES ANDERSON PALETTE GENERATOR, 2018, Retrieved from the Internet <URL:https://cran.r-project.org/package=wesanderson>
RICE ET AL.: "EMBOSS: The European Molecular Biology Open Software Suite", TRENDS GENET., vol. 16, 2000, pages 276 - 277, XP004200114, DOI: 10.1016/S0168-9525(00)02024-2
ROMANOS ET AL., YEAST, vol. 8, 1992, pages 423 - 488
ROTH, J. S.LOSTY, T.WIERBICKI, E.: "Assay of proteolytic enzyme activity using a C-labeled hemoglobin", ANALYTICAL BIOCHEMISTRY, vol. 42, no. 1, July 1971 (1971-07-01), pages 214 - 221, XP024824818, DOI: 10.1016/0003-2697(71)90029-7
S. AMIDON ET AL., AAPS PHARMSCITECH., vol. 16, no. 4, August 2015 (2015-08-01), pages 731 - 741
SAMBOL, S. P.MERRIGAN, M. M.JOHNSON, S.GERDING, D. N.: "Infection of Hamsters with Epidemiologically Important Strains of Clostridium difficile", THE JOURNAL OF INFECTIOUS DISEASES, vol. 183, no. 12, 15 June 2001 (2001-06-15), pages 1760 - 1766
SORG, J. A.SONENSHEIN, A. L.: "Bile salts and glycine as cogerminants for Clostridium difficile spores", JOURNAL OF BACTERIOLOGY, vol. 190, no. 7, 2008, pages 2505 - 2512, XP002636062, DOI: 10.1128/JB.01765-07
SORG, J. A.SONENSHEIN, A. L.: "Chenodeoxycholate is an inhibitor of Clostridium difficile spore germination", JOURNAL OF BACTERIOLOGY, vol. 191, no. 3, 2009, pages 1115 - 1117, XP055027727, DOI: 10.1128/JB.01260-08
STAGGERS, J. E.HERNELL, O.STAFFORD, R. J.CAREY, M. C.: "Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings", BIOCHEMISTRY, vol. 29, no. 8, 1990, pages 2028 - 2040, Retrieved from the Internet <URL:https://doi.org/10.1021/bi00460a011>
SUN ET AL.: "Mouse Relapse Model of Clostridium difficile Infection", INFECTION AND IMMUNITY, vol. 79, no. 7, 2011, pages 2856 - 2864, XP055191408, DOI: 10.1128/IAI.01336-10
SURAWICZ, C. M.MCFARLAND, L. V.GREENBERG, R. N.RUBIN, M.FEKETY, R.MULLIGAN, M. E.GARCIA, R. J.BRANDMARKER, S.BOWEN, K.BORJAL, D.: "The search for a better treatment for recurrent Clostridium difficile disease: use of high-dose vancomycin combined with Saccharomyces boulardii", CLIN INFECT DIS., vol. 31, no. 4, October 2000 (2000-10-01), pages 1012 - 7, XP002253947, DOI: 10.1086/318130
TANAKA, H.HASHIBA, H.KOK, J.MIERAU, I.: "Bile salt hydrolase of Bifidobacterium longum-biochemical and genetic characterization", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 66, no. 6, 2000, pages 2502 - 2512, XP002346523, Retrieved from the Internet <URL:https://doi.Org/10.1128/aem.66.6.2502-2512.2000> DOI: 10.1128/AEM.66.6.2502-2512.2000
THERIOT C.M.KOENIGSKNECHT M.J.CARLSON P.E. ET AL.: "Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection", NAT COMMUN, vol. 5, 2014, pages 3114
TOOTHAKER, R. D.ELMER, G. W.: "Prevention of clindamycin-induced mortality in hamsters by Saccharmoyces boulardii", 1984, AMERICAN SOCIETY FOR MICROBIOLOGY
VANEGAS, K.G.LEHKA, B.J.MORTENSEN, U.H.: "SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae", MICROB CELL FACT, vol. 16, 2017, pages 25, Retrieved from the Internet <URL:https://doi.org/10.1186/sl2934-017-0632-x>
WENTZ, A. E.SHUSTA, E. V.: "Enhanced Secretion of Heterologous Proteins from Yeast by Overexpression of Ribosomal Subunit RPPO", BIOTECHNOLOGY PROGRESS, vol. 24, no. 3, 2008, pages 748 - 756, Retrieved from the Internet <URL:https://doi.org/10.1021/bp070345m>
WICKHAM, H., GGPLOT2: ELEGANT GRAPHICS FOR DATA ANALYSIS, 2016, Retrieved from the Internet <URL:https://ggplot2.tidvverse.org>
YOON, S.YU, J.MCDOWELL, A.KIM, S. H.YOU, H. J.KO, G.: "Bile salt hydrolase-mediated inhibitory effect of Bacteroides ovatus on growth of C. difficile", JOURNAL OF MICROBIOLOGY, vol. 55, no. 11, 2017, pages 892 - 899, Retrieved from the Internet <URL:https://doi.org/10.1007/sl2275-017-7340-4>

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